Automated performance of polymerase chain reaction

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DESCRIPTION

Procedure to carry out the polymerase chain reaction.

Containers to carry out the polymerase chain reaction.

The invention belongs to the field of computer controlled instruments for carrying out the polymerase chain reaction (hereinafter PCR, from its acronym in English Polymerase Chain Reaction). More particularly, the invention pertains to automated instruments that can perform the polymerase chain reaction simultaneously on many samples with a high degree or precision in the results obtained for each sample. This high precision provides the ability, among other things, to perform so-called "quantitative PCR".

To amplify DNA (deoxyribonucleic acid) using the PCR process, it is necessary to run a specially constituted liquid reaction mixture through a PCR protocol including several incubation periods at different temperatures. The reaction mixture is composed of several components such as the DNA to be amplified and two primers selected at least in a predetermined way to be sufficiently complementary with the DNA sample to allow to create extension products of the DNA to be amplified. The reaction mixture includes various enzymes and / or other reagents, as well as various deoxyribonucleoside triphosphate such as dATP, dCTP, dGTP, and dTTP. Generally, primers are oligonucleotides capable of acting as a synthesis initiation point when placed under conditions in which the synthesis of a primer extension product that is complementary to the nucleic acid chain is induced, that is, in presence of nucleotides and inducing agents such as thermostable DNA polymerase at a suitable temperature and pH.

The polymerase chain reaction (PCR) has proven to be a phenomenally successful technology in genetic analysis, primarily because it is very simple and requires relatively low-cost instrumentation. A key to PCR is the concept of thermocycles: alternative DNA fusion steps, short annealing of primers to obtain single resulting strands, and the extension of these primers to make new copies of the DNA double strand. In thermocycling, the PCR reaction mix is ​​repeatedly passed through high temperatures (> 90 ° C) to melt the DNA, at low temperatures (40 ° C to 70 ° C) for primer extension and hybridization. The first commercial system to perform the thermal cycling required in the polymerase chain reaction, the "PerkinElmer Cetus DNA Thermal Cycler", was introduced in 1987.

Applications of PCR technology are now moving from basic research to applications where large numbers of similar amplifications are routinely developed. These areas include diagnostic research, biopharmaceutical development, genetic analysis, and environmental testing. Users in these areas would benefit from a high throughput PCR system that would provide the user with high productivity, fast cycle time, and reproducible results. Users in these areas must ensure reproducibility from sample to sample, run to run, lab to lab, and instrument to instrument.

For example, the process for the physical map in the human genome project can be greatly simplified using identified sequence sites. An SSI is a short and unique sequence easily amplified by PCR and identifying a location on the chromosome. Searching for such genome mapping sites requires amplifying large numbers of samples in a short time with protocols that can be reproducible worldwide.

As the number of PCR samples increases, it becomes more important to integrate amplification with sample preparation and post-amplification analysis. Sample containers should allow not only rapid thermal cycling but also automated handling for operations such as solvent extraction and centrifugation. Containers should operate consistently at low volumes to reduce reagent costs.

Generally, PCR temperature cycling involves at least two incubations at different temperatures. One of these incubations is for primer annealing and primer extension catalytic writing. The other incubation is for denaturation, that is, separation of the double-stranded extension products into single-stranded standards for use in the next hybridization and incubation interval of the extension. Details of the polymerase chain relationship, the temperature cycling and reaction conditions necessary for PCR as well as the various reagents and enzymes required to perform the reaction are described in US Patents 4,683,202, 4,683,195, publication EPO 258,017 and 4,889,818 (Tqa polymerase enzyme patent) and all other PCR patents assigned to Cetus Corporation.

The purpose of the polymerase chain reaction is to make a large volume of DNA that is identical to a small volume initially supplied in the DNA "seed". The relationship involves copying strands of DNA and then using the copies to generate other copies in subsequent cycles. Under ideal conditions, each cycle will double the amount of DNA present thereby producing a geometric progression in the copy volume of the "target" or "seed" DNA strands present in the reaction mixture.

A typical cycle of PCR temperatures requires that the reaction mix be accurately held at each incubation temperature for a prescribed time and that identical or similar cycles be repeated many times.

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A typical PCR program begins with a sample temperature of 94 ° C held for 30 seconds to denature the reaction mix. Then, the temperature of the reaction mixture is lowered to 37 ° C and held for one minute to allow the first hybridization. Next, the temperature of the reaction mixture is raised to a temperature in the range of 50 ° C to 72 ° C where it is maintained for two minutes to promote the synthesis of the extension products. This completes a cycle. The next PCR cycle then begins by raising the temperature of the reaction mixture again to 94 ° C for the separation of the chains of the extension products formed in the previous cycle (denaturation). Typically, the cycle is repeated 25 to 30 times.

Generally, it is desirable to change the temperature of the sample to the next temperature in the cycle as quickly as possible for a number of reasons. First, the chemical reaction has an optimal temperature for each of its stages. Thus, less time spent at non-optimal temperatures means better chemical results are obtained. Another reason is that the reaction mixture needs to be kept at each incubation temperature for a minimum time after reaching that incubation temperature. These minimum incubation times establish the minimum or "base" time it takes to complete a cycle. Any transition time between the incubation temperatures of the sample is time that is added to this minimum cycle time. Since the number of cycles is considerably large, this additional time unnecessarily lengthens the total time required to complete amplification.

In some earlier automated PCR instruments, the reaction mix was stored in a disposable plastic tube and closed with a lid. A typical sample volume for such tubes was approximately 100 microliters. Typically, such instruments used many such tubes filled with sample DNA and reaction mix inserted into holes called sample wells in a metal block. To perform the PCR process, the temperature of the metal block was controlled according to the prescribed temperatures and times specified by the user in a PCR protocol file. A computer and associated electronics then controlled the temperature of the metal block in accordance with the data supplied by the user in the PCR protocol file that defines times, temperatures and the number of cycles, etc. As the temperature of the metal block changed, the samples in the various tubes followed them with similar temperature changes.

However, in these prior art instruments not all samples experienced exactly the same temperature cycle. In these prior art PCR instruments, errors were generated in the sample temperature due to the non-uniformity of the temperature from site to site within the metal sample block, that is, there were temperature gradients within the metal block and hence that some samples had different temperatures than other samples at particular times in the cycle. Also, there were delays in heat transfer from the sample block to the sample, and the delays were not the same for all samples. In order to perform the PCR process successfully and efficiently, and to allow so-called "quantitative" PCR, these lag times and temperature errors must be greatly minimized.

The problems of minimizing delay times for heat transfer to and from the liquid sample and minimizing temperature errors due to temperature gradients or non-uniformity in temperature at various points on the metal block become particularly acute when the size the region containing the mixtures becomes large. It is a highly desirable attribute for a PCR instrument to have a metal block that is large enough to accommodate 96 sample tubes arranged in the format of an industry standard microtiter plate.

The microtiter plate is a widely used medium for the handling, processing and analysis of large numbers of small samples in the fields of biochemistry and biotechnology. Typically, a microtiter plate is a 9.2 cm (3 5/8 inch) wide and 12.7 cm (5 inch) long tray containing 96 identical sample wells in an 8 by 12 rectangular array. wells on centers at 9 millimeters. A wide variety of equipment is available for automating the handling, processing and analysis of samples in this standard microtiter plate format.

Microtiter plates are generally made from injection molded or vacuum formed plastic and are inexpensive and considered disposable. Being disposable is a highly desirable feature due to the legal liability for cross contamination and the difficulty of cleaning and drying the microtiter plates after use.

It is therefore a highly desirable characteristic for a PCR instrument to be able to perform the PCR reaction on up to 96 samples simultaneously, said samples being arranged in a microtiter plate format.

Naturally, the size of the metal block that is necessary to heat and cool 96 samples in an 8 x 12 well array with centers at 9 mm is considerably large. That large area block creates multiple engineering challenges for designing a PCR instrument that is capable of heating and cooling the block very quickly in a temperature range generally 0 to 100 ° C with very little tolerance for temperature variations. between samples. These problems come from various sources. First, the large thermal mass of the block makes it difficult to change the temperature of the block up and down the operating range very quickly. Second, the need to attach the block to various external devices such as manifolds for the supply and withdrawal of the cooling liquid, fixing points of the block support and other associated peripheral equipment, creates the potential for the existence of temperature gradients. across the block that exceed tolerable limits.

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There are also numerous other conflicts in the design requirements of a thermal cycling system for the automated performance of the PCR reaction or other reactions that require fast and accurate temperature cycling on a large number of samples. For example, to change the temperature of a metal block rapidly, a large amount of heat must be added to it or removed from the sample block in a short period of time. Heat can be added with an electrical resistance heater or by circulating a heating fluid in contact with the block. The heat can be removed quickly by circulating cooling fluid in contact with the block. However, it is apparently impossible to add or remove large amounts of heat rapidly in a metal block by these means without producing large temperature differences from site to site of the block and thus forming temperature gradients that can result in non-uniformity of the block. temperature between samples.

Even after the process of adding or removing heat is completed, temperature gradients can persist for a time roughly proportional to the square of the distance that the heat stored at various points in the block must travel to the coldest regions to eliminate the temperature gradient. . Thus, by making the metal block larger to accommodate more samples, the time it takes to decrease the temperature gradients in the block after a change in temperature produces temperature gradients spread across the larger dimensions of the block, can be as high as be markedly older. This makes it increasingly difficult to cycle through the temperature cycles of the sample block rapidly while maintaining precise temperature uniformity among all samples.

Due to the time required to dissipate temperature gradients, an important need has arisen in the design of a high-throughput PCR instrument to prevent the creation of temperature gradients that span long distances in the block. Another need is to avoid, as much as possible, the need for heat to pass through mechanical boundaries between the metal parts and other peripheral equipment attached to the block. It is difficult to join metal parts in a way that ensures uniform high thermal conductivity at any point throughout the joint. Non-uniformities in thermal conductivity will generate unwanted temperature gradients.

The invention provides a method for performing polymerase chain reaction (PCR) which comprises providing a thermocycling apparatus comprising a sample block containing one or more sample wells, one or more capped reaction tubes arranged in one or more more sample wells and a heated roller to contact the caps of the one or more reaction tubes, each tube comprising a PCR reaction mix, subjecting the reaction mixture or mixtures to a PCR protocol comprising a plurality of repeated cycles by cycling the temperature of the sample block, each cycle comprising at least one primer extension temperature and one denaturation temperature, maintaining said roller heated to a temperature of 94 ° C to 110 ° C and remaining in contact with the caps of one or more tubes during the PCR protocol, thus avoiding water condensation on the lower parts of the covers.

Accordingly, the contents of the invention contemplate a new method of preventing solvent loss from reaction mixtures when samples are being incubated at temperatures near their boiling point. A heated roller covers the tops of the sample tubes and is in contact with an individual cap that provides a gas tight seal for each sample tube. The heat from the roller heats the tops of each sample tube and the cap to a temperature above the dew point so that condensation and reflux do not occur within any of the sample tubes. Condensation represents a relatively large heat transfer since an amount of heat equal to the heat of vaporization occurs when water vapor condenses. This could cause large temperature variations from sample to sample if condensation did not occur uniformly. The heated roller prevents any condensation from occurring in any sample tube thus minimizing this source of potential temperature errors. Using the heated roller also reduces reagent consumption.

In addition, the heated roller provides a downward force for each sample tube that exceeds an experimentally determined minimum downward force necessary to keep all sample tubes firmly pressed into the temperature-controlled sample block to establish and maintain a thermal conductance of block to tube for each tube. This uniformity of thermal conductance is established independently of tube-to-tube variations in length, diameter, angle, or other dimensional errors that would otherwise cause some of the sample tubes to fit more snugly in their corresponding sample wells than other sample tubes.

The heated roller preferably softens the plastic in each cap but does not totally destroy the elasticity of the caps. In this way, a minimum threshold downward force is successfully applied to each tube rather than differences in tube height from tube to tube.

The PCR instrument described in this document reduces cycle times by a factor of 2 or greater and reduces reagent cost by adapting PCR volumes up to 20 uh while still being compatible with the 0.5 microcentrifuge tube industry standard. ml. The present invention will now be further described, by way of example only, with reference to the accompanying drawings, in which:

Figure 1 is a block diagram of the thermal cycle that can be used in accordance with the teachings of the invention.

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Figure 2 is a plan view of the sample block in accordance with the teachings of intent.

Figure 3 is a side elevation view of the sample block showing the rapid cooling and control channels.

Figures 4 and 5 are the end elevation views of the sample block.

Figure 6 is a sectional view of the sample block taken along line 6-6 'in Figure 2.

Figure 7 is a sectional view of the sample block taken along line 7-7 'in Figure 2.

Figure 8 is a sectional view of the sample block taken along line 8-8 'in Figure 2.

Figure 9 is a sectional elevation view of the sample block frame after mounting of the three-zone film heater and block holder.

Figure 10 is a line voltage graph illustrating the way to control power to the three zone film heater.

Figure 11 is a temperature graph showing a typical PCR protocol of incubation at three temperatures.

Figure 12 is a sectional view of the sample block illustrating the local zone concept.

Figure 13 is a plan view of the three zone heater.

Figure 14 is a graph of sample temperature over time illustrating the effect of the time constant of a sample tube whose seating force F is too low.

Figure 15 is a sectional view of the sample tube and cap seated on the sample block.

Figure 16A is a graph of the impulse response of an RC circuit.

Figure 16B is a graph of a drive pulse.

Figure 16C is a graph illustrating how the convolution of the thermal impulse response and the temperature history of the block gives the calculated sample temperature.

Figure 16D illustrates the electrical analogy of the sample block / sample tube system thermal response.

Figure 17 illustrates how the calculated temperatures of the six different samples all converge to a target temperature in the neighborhood of 0.5 ° C each when the proportionality constants for the equations used in the three-zone heater controller are properly adjusted. .

Figure 18 is a graph illustrating how the denaturation target temperature affects the amount of DNA generated.

Figure 19 is a sectional view of the slide cover and heated base plate.

Figure 20 is a perspective view of the slide cover, sample block, and knob used to lower the heated plate.

Figure 21A is a sectional view of the mounting of one embodiment of the frame, retainer, sample tube, and cap when seated on the sample block.

Figure 21B is a sectional view of the preferred embodiment of the frame, retainer, sample tube, and cap when seated on the sample block.

Figure 22 is a top plan view of the disposable, plastic microtiter plate frame.

Figure 23 is a bottom plan view of the frame.

Figure 24 is an end elevation view of the frame.

Figure 25 is an elevation view at the other end of the frame.

Figure 26 is a sectional view of the frame taken along line 26-26 'in Figure 22.

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Figure 27 is a sectional view of the frame taken along line 27-27 'in Figure 22.

Figure 28 is an edge elevation view and partial section of the frame.

Figure 29 is a sectional view of the preferred sample tube.

Figure 30 is a sectional view of the top of the sample tube.

Figure 31 is an elevational view of a portion of the cap band.

Figure 32 is a top view and a part of the band of caps.

Figure 33 is a top plan view of the disposable plastic retainer portion of the 96-well microtiter tray.

Figure 34 is a side elevation view with a partial section of the retainer.

Figure 35 is an end elevational view of the retainer.

Figure 36 is a sectional view of the retainer taken along line 36-36 'in Figure 33.

Figure 37 is a sectional view of the retainer taken along line 37-37 'in Figure 33.

Figure 38 is a plan view of the disposable plastic support base of the 96-well microtiter tray.

Figure 39 is a bottom plan view of the base.

Figure 40 is a side elevation view of the base.

Figure 41 is an end elevation view of the base.

Figure 42 is a sectional view of the support base taken along line 42-42 'in Figure 38.

Figure 43 is a sectional view of the support base taken along line 43-43 'in Figure 38.

Figure 44 is a sectional view of the base taken along line 44-44 'in Figure 38.

Figure 45 is a perspective view of the extension of the disposable plastic elements that comprise the microtiter plate with some sample tubes and caps in position.

Figure 46 is a diagram of the refrigeration control system 24 of Figure 1.

Figures 47A and 47B are block diagrams of control electronics in accordance with the teachings of the invention.

Figure 48 is a schematic of a typical zener temperature detector.

Figure 49 is a diagram of the timeline of a typical sample period.

Figure 50 is an elevational sectional view of a tall, thin-walled sample tube sold under the trademark MAXIAMP.

Fig. 51 is a graph showing the different response in time between a thin-walled sample tube and earlier run thick-walled tubes.

Figure 52 is a plan view of the sample tube and cap.

Figures 53 and 54 are flow charts of the initial test sequence.

Referring to Figure 1, it shows a block diagram of the main components of the system of an embodiment of a computer-driven instrument for performing PCR. The sample mixes that include the DNA or RNA to be amplified are placed in the programmed temperature sample block 12 and covered with the heated cover 14.

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A user supplies the data of the parameters that define the time and temperature of the desired protocol of the PCR through a terminal 16 that includes a keyboard and a screen. The keyboard and display are coupled via bus 18 to a control computer 20 (hereinafter referred to as the central processing unit or CPU). This central processing unit 20 includes the memory that stores the control program described later, the data that defines the desired protocol of the PCR and certain calibration constants described later. The control program causes the CPU 20 to control the temperature cycles of the sample block 12 and implement a user interface that provides certain screens to the user and which receives the data entered by the user through the keyboard of the terminal 16.

In the preferred embodiment, the central processing unit 20 is made specifically for the application.

A block diagram of the electronics will be discussed in more detail later. In alternative embodiments, the central processing unit 20 and associated peripheral electronics for controlling the various heaters and other electromechanical systems of the instrument and reading various detectors can be any general purpose computer such as a suitably programmed personal computer or microcomputer.

The samples 10 are stored in capped disposable tubes which sit in the sample block 12 and are thermally isolated from the ambient air by the heated cover 14 which is brought into contact with the plastic disposable tray to be described later to form a heated, closed box in which the sample tubes reside. The heated cover serves, among other things, to reduce unwanted heat transfers to and from the sample mixture by evaporation, condensation and reflux within the sample tubes. It also reduces the opportunity for cross contamination by keeping the insides of the caps dry and thereby preventing the formation of vapors when the tubes are uncapped. The heated cover is in contact with the caps of the sample tube and keeps them heated to a temperature of approximately 104 ° C or above the dew points of the various components of the reaction mixture.

The central processing unit 20 includes the appropriate electronics to sense the temperature of the heated cover 14 and control electrical resistance heaters to maintain the cover 14 at a predetermined temperature. Here the sensing of the temperature of the heated cover 14 and the control of the resistance heaters is carried out via a temperature sensor (not shown) and the bus 22.

A coolant control system 24 continuously circulates a cold coolant such as a mixture of water and automobile antifreeze through the control cooling channels (not shown) in the sample block 12 through the inlet tubes. 26 and outlet tube 28. The coolant control system 24 also controls the high volume flow of the rapid cooling fluid through the circulation paths (not shown) in the sample block 12. The rapid cooling channels are used to rapidly change the temperature of the tank. sample block 12 pumping large volumes of cooled liquid refrigerant through the block at a relatively high flow rate. The rapid cooling cooling liquid enters the sample block 12 through tube 30 and exits the sample block through tube 32. The details of the cooling control system are shown in Figure 46. The cooling control system Refrigeration will be discussed more fully later in the description of the electronics and the control system program.

Typically, the coolant used to cool the sample block 12 consists primarily of a mixture of water and glycol. The refrigerant liquid is cooled in a heat exchanger 34 that receives the refrigerant liquid, from which the heat has been extracted, from the sample block 12 through the tube 36. The heat exchanger 34 receives the refrigerant through the inlet tube 38 compressed liquid freon from a refrigeration unit 40. This refrigeration unit 40 includes a compressor (not shown), a fan 42, and a finned tube heat radiator 44. The refrigeration unit 40 compresses the freon gas received from the heat exchanger 34 through the tube 46. The freon Gaseous is cooled and condensed to liquid in finned tube condenser 44. The pressure of the liquid freon is maintained above its vapor pressure in the finned condenser tube through a capillary tube flow restrictor 47. The outlet of this capillary tube is coupled to the inlet of the heat exchanger 34 by means of the tube 38. In the heat exchanger, the pressure of the freon is allowed to drop below the vapor pressure of the freon, and the freon expands. In this expansion process, heat is absorbed from the heated coolant flowing through the heat exchanger and this heat is transferred to the freon thereby causing the freon to boil. The heated freon is then decompressed from the heat exchanger through tube 46 and again circulated through finned tube condenser 44. Fan 42 blows air through finned condenser tube 44 to cause heat in the freon in tube 46 to exchange with ambient air. As symbolized by arrows 48. Refrigeration unit 40 should be capable of extracting 400 W of heat at 30 ° C and 100 watts of heat at 10 ° C from the coolant to maintain rapid temperature cycling in accordance with the teachings. of the invention.

In the preferred embodiment, the apparatus of Figure 1 is contained within the housing (not shown). The heat 48 expelled to the ambient air is kept within the shell to aid the evaporation of any condensation that may occur in the various tubes that carry the cooled liquid refrigerant or Freon from one location to another. This condensation can lead to corrosion on the metals used in the construction of the unit or on the electronic circuits and must be removed. By expelling the heat 48 into the enclosure it helps to evaporate any condensation and prevent corrosion.

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After exchanging its heat with the freon, the liquid refrigerant is withdrawn from the heat exchanger 34 through the tube 50 and reintroduced into the refrigeration control system where it is regulated as needed to the sample block during parts of the cycle. fast cooling PCR defined by user input via terminal 16.

As stated above, the PCR protocol involves incubation at at least two different temperatures and often three different temperatures. A typical PCR cycle is shown in Figure 11 with a denaturation incubation 170 performed at a temperature close to 94 ° C, a hybridization incubation 122 performed at a temperature close to room temperature (25 ° C-37 ° C ) and an extension incubation 174 carried out at a temperature close to 50 ° C. These temperatures are substantially different, and therefore means must be provided to change the temperature of the reaction mixture of all samples rapidly from one temperature to another. The blast chill system is the means by which the temperature of the sample block 12 is rapidly lowered from the high incubation temperature of denaturation to the low temperatures of incubation of hybridization and extension. Typically the coolant temperature is in the range of 10-20 ° C. When the refrigerant is at 20 ° C it can pump about 400 W of heat from the sample block. Typically the dimensions of the fast cooling channel, the coolant temperature and the coolant flow rate are adjusted so that a cooling peak of 5 ° -6 ° C per second can be achieved in the vicinity of the high end of the operating range (100 ° C) and an average cooling rate of 2.5 ° C per second is achieved by lowering the temperature of the sample block from 94 ° C to 37 ° C.

The gradual cooling system, in some embodiments, can also be used to maintain the temperature of the sample block at or near the incubation temperature as well. However, in the preferred embodiment, small temperature changes of the sample block 12 in the downward direction to maintain the incubation target temperature are accomplished through the control refrigeration system.

As shown in figure 46, a pump 41 constantly pumps refrigerant from a reservoir / filter 39 (130 milliliters capacity) through a 1.3 cm (1/2 ”) pipe and pumps it through the pipe. 1/2 ”(1.3 cm) to bypass intersection 47. Pump 41 supplies refrigerant to line 45 at a constant flow rate of 1-1.3 gallons per minute (3.8-4.9 L / minute). minute). At intersection 47, a portion of the flow from tube 45 is diverted as constant flow through control cooling channels 49, another portion of flow from tube 45 is diverted through flow restrictor 51 to outlet tube 38. The flow restrictor 51 maintains sufficient pressure in the system so that there is positive pressure at the inlet 53 of a solenoid actuated two-state valve 55 under the control of the CPU 20 via the bus 54. When rapid cooling is desired to perform a rapid drop in temperature, CPU 20 causes solenoid-operated valve 55 to open to allow the flow of refrigerant through rapid cooling channels 57. There are 8 rapid cooling channels in a continuous manner. that the flow rate through each rapid cooling channel is around 0.5 l / min. The flow rate through the control cooling channels is much lower due to the very small sectional area of ​​the channels.

The control refrigeration system provides a small constant flow of cold refrigerant through the control refrigeration channels 49 in the sample block 12. This results in a small and constant heat loss from the sample block 12 which is compensated by the multi-zone heater 156 which is thermally coupled to sample block 12 during incubation segments in which the temperature of the sample block is to be maintained at a stable value. The small constant heat loss produced by the control cooling flow allows the control system to implement proportional temperature control both up and down for low temperatures. This means that heating and cooling at small, predictable, and controlled rates is available in the temperature control system to correct for block temperature errors and make the block temperature closely follow the user-entered PCR temperature profile. The alternative would be to cut power to the film heater and allow the sample block to cool by delivering heat to the environment by radiation and convection when the temperature of the block becomes too high. This would be too slow and too unpredictable to meet the tight temperature control specifications for a few cycles of quantitative PCR.

The multi-zone heater 156 is controlled by the CPU 20 via the bus 52 of Figure 1 and is the means by which the temperature of the sample block 12 increases rapidly at the highest incubation temperatures from the lowest incubation temperatures. low and is the medium through which control cooling is compensated and temperature errors are corrected in the upward direction during control and monitoring of temperatures during incubations.

In alternative embodiments, the control cooling can be supplied by other means such as the use of a cooling fan and cooling foils formed in the metal of the sample block, Peltier joints, or constantly circulating tap water. Care must however be taken in these alternative embodiments to ensure that temperature gradients are not created in the sample block which would cause the temperature of some samples to differ from the temperature of other samples thereby possibly producing different amplification results in the sample. PCR in some sample tubes with respect to others. In the preferred embodiment, the control cooling is proportional to the difference between the block temperature and the coolant temperature.

The CPU 20 controls the temperature of the sample block 12 by sensing the temperature of the metal of the sample block through the temperature detector 21 and bus 52 of Figure 1 and by sensing the temperature.

ES 2 318 232 T3 of the refrigerant liquid circulating through bus 54 and a temperature sensor in the refrigeration control system. The coolant temperature sensor is shown at 61 in Figure 46. The CPU also detects the internal ambient air temperature within the system housing through an ambient air temperature sensor 56 in Figure 1. Additionally, the CPU 20 detects the line voltage of the power input on line 58 through the detector symbolized at 63. All this data together with the data entered by the user to define the desired PCR protocol such as temperatures and times Targets for incubations are used by the control program described in detail below. This control program calculates the amount of power to apply to the various zones of the multi-zone film heater 156 of the sample block via bus 52 and generates a refrigeration control signal to open or close the solenoid-operated valve 55 at cooling control system 24 via bus 54 to make the sample block temperature follow the PCR protocol defined by user input.

Referring to Figure 2, a top view of the sample block 12 is shown. The purpose of the sample block 12 is to provide mechanical support and a heat exchange element for an array of thin-walled sample tubes where heat can be exchanged between the liquid sample in each sample tube and the cooling liquid circulating in the channels of the control cooling and the rapid cooling formed in the sample block 12. Additionally, it is the function of the sample block 12 to provide its heat exchange function without creating large temperature gradients between the various sample wells such that all sample mixes in the array undergo the same PCR cycle even though they are spatially separated. It is a general objective of the PCR instrument described herein to provide a very tight temperature control for the temperature of the liquid sample of a variety of samples so that the temperature of any liquid sample does not vary appreciably (approximately plus or minus 0.5 ° C) relative to the temperature of any other liquid sample in another well at any point in the PCR cycle.

There is an emerging branch of PCR technology called "quantitative" PCR. In this technology, the goal is to perform PCR amplification as precisely as possible to make the amount of target DNA exactly double each cycle. Exact doubling on each cycle is difficult or impossible to achieve but tight temperature control helps.

There are many sources of errors that can cause the PCR cycle to fail to exactly duplicate the amount of target DNA (hereinafter DNA should be understood as also referring to RNA) during one cycle. For example, in some PCR amplifications, the process begins with a single target DNA cell. An error that can easily occur occurs when this single cell attaches to the wall of the sample tube and does not amplify in several of the first few cycles.

Another type of error is the entry of foreign nuclease into the reaction mixture which attacks the "foreign" target DNA. All cells have some non-specific nuclease that attacks foreign DNA that is loose in the cell. When this occurs, it interferes with or stops the replication process. Thus, if a drop of saliva or a dandruff particle or material from another sample mixture were inadvertently introduced into a sample mixture, the nuclease materials in these cells could attack the target DNA and cause an error in the amplification process. It is highly desirable to totally eliminate such sources of cross contamination.

Another source of error is inaccurate control over the temperature of the sample mixture, between several of the multiple different samples. For example, if all samples are not precisely controlled to have the proper hybridization temperature (a user-selected temperature typically in the 50 to 60 ° C range) for the length of incubation, certain forms of DNA will not spread. adequately. This occurs because the primers used in the extension process hybridize to the wrong DNA if the temperature is too low. If the hybridization temperature is too high, the primers will not hybridize to the target DNA at all.

The consequences of performing the PCR amplification process inaccurately can be easily imagined when the PCR amplification is part of diagnostic tests such as the presence of HIV antibodies, hepatitis or the presence of genetic diseases such as sickle cell anemia, etc. A false positive or false negative result on such diagnostic tests can have disastrous personal and legal consequences. Consequently, it is an objective for the design of the PCR instrument described herein to eliminate as many of these sources of possible errors as possible such as cross-contamination or poor temperature control while providing an instrument that is compatible with the format. of the industry standard 96-well microtiter plate. The instrument should perform PCR quickly in a flexible way with a simple user interface.

In the preferred embodiment, the sample block 12 is machined from a solid block of relatively pure yet corrosion resistant aluminum such as 6061 aluminum alloy. Machining the structure of the block from a solid block of aluminum results in a more thermally homogeneous structure. Cast aluminum structures tend not to be as thermally homogeneous as necessary to meet the very narrow desired temperature control specifications.

The sample block 12 is capable of rapid temperature changes because the thermal mass of the block is kept low. This is accomplished by the formation in the block of many cooling paths, sample wells, grooves, and other threaded or unthreaded holes. Some of these holes are used to fix the block to supports and to fix external devices such as collectors and pour pans there.

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To better appreciate the “honeycomb” nature of the structure of the sample block, we refer the reader simultaneously to Figure 2 which shows the block seen in plan as well as Figures 3 to 8 which show elevations and sectional views of the sample block strategically located. For example, Figure 3 is an elevation showing the positions of the cooling channel seen from the strategic point of line 3-3 'in Figure 2. The elevation of the sample block 12, viewed from the opposite side, is identical. Figure 4 is an edge elevation of sample block 12 viewed from the perspective of line 4-4 'in Figure 2. Figure 5 is an end elevation of sample block 12 viewed from the perspective of line 5 -5 'in Figure 2. Figure 6 is a section of sample block 12 taken along section line 6-6' in Figure 2. Figure 7 is a section of sample block 12 taken along section line 7-7 'in Figure 2. Figure 8 is a section of sample block 12 taken along section line 8 -8 'in figure 2.

The upper surface of sample block 12 is drilled with an 8 x 12 array of conical sample wells of which two wells 66 and 68 are typical. The conical configuration of each sample well is best seen in Figure 8. The walls of each sample well are drilled at a 17 ° angle to match the angle of the conical section of each sample tube. This is done by drilling a pilot hole that has a diameter D_w in Figure 8. A 17 ° countersink is then used to form the tapered walls 67.

The bottom of each sample well includes a sump 70 that has a depth that exceeds the penetration depth of the tip of the sample tube. Sump 70 is created by the pilot hole and provides a small open space below the sample tube when the sample tube sits in the corresponding sample well. This sump provides a space for liquids such as condensation that forms on the walls of the well to reside in it without interfering with the tight fit of each sample tube to the walls of the sample well. This tight fit is necessary to ensure that the thermal conductivity between the well wall and the liquid sample is uniform and high in each sample tube. Any contamination in a well that causes a loose fit for a tube destroys this uniformity of thermal conductivity throughout the matrix. This is, because the liquid is substantially incompressible to the pressures involved in the seating of the sample tubes in the sample wells, if the sump 70 were not present, the presence of the liquid at the bottom of the sample well could prevent the full seat of the sample tube in its sample well. Furthermore, sump 70 provides space for a gaseous phase of any liquid residing in sump 70 to expand during high temperature incubations such that the large forces of such expansion that would be present if the sump were not present 70, do not apply to the sample tube to press the tube away from contact with the sample well.

It was found experimentally that it is important that each sample tube is glued with its corresponding sample well and that a certain minimum level of force is applied to each sample tube to maintain thermal conductivity between the walls of the sample well and the mixture of samples. uniform reaction throughout the matrix. This minimum level of seating force is shown as a force vector F in Figure 15 and is a key factor in preventing the thermal conductivity through the walls of a sample tube from being different than the thermal conductivity through the walls. walls of another sample tube located elsewhere in the block. The minimum seating force level F is 30 grams and the preferred force level is between that and 100 grams.

The sample well array is essentially completely surrounded by a slot 78, best seen in Figures 2, 6, and 8, which has two functions. The main function is to reduce the thermal conductivity from the central area of ​​the sample block to the edge of the block. Slot 78 extends approximately 2/3 the thickness of the sample block. This groove minimizes the effects of the thermal gradients inevitably produced by the necessary mechanical connections with the block of fixing bolts, valves, etc. A secondary function is to extract thermal mass from sample block 12 to allow the temperature of sample block 12 to vary more rapidly and to simulate a row of wells in the edge region called the "guard band." The amount of metal drawn by the portion of the groove 78 between points 80 and 82 in Figure 2 is designed to be substantially the same as the amount of metal drawn by the adjacent column of eight sample wells 83 through 90. The purpose of this is equating the thermal mass of the protection band with the thermal mass of the adjacent “local zone”, a term that we will explain more fully later.

Referring specifically to Figures 3, 6 and 8, the number and relative positions of various control cooling and rapid cooling channels that are formed in the metal of sample block 12 are shown. There are nine control cooling channels marked with the reference numbers 91 to 99. In the same way, there are eight rapid cooling channels marked with reference numbers 100 to 107.

Each of these rapid cooling and control cooling channels is cooled drilled through the aluminum of the sample block. The cooling drilling process is well known and provides the ability to drill a long and very straight hole that is as close as possible to the bottom surface 110 of the sample block 12. Since the cooling drilling process drills a straight hole, this process is preferred to prevent any of the rapid cooling or control cooling channels from being bypassed during the drilling process and penetrating the bottom surface 110 of the sample block or into otherwise alter their position relative to the other cooling channel. Such displacements could cause undesirable temperature gradients altering the "local equilibrium" and "local symmetry" of the local zones. These concepts are explained later, but by now the reader should understand that these notions and the structures that implement them are

ES 2 318 232 T3 key to fast temperature cycling of up to 96 samples without creating excessive temperature errors between different sample wells.

The control cooling channels 91 through 99 are covered with silicone rubber in the preferred embodiment to reduce thermal conductivity through the walls of the control cooling channels. It is preferred to decrease the thermal conductivity through the channel walls in the control cooling channels to prevent too rapid temperature changes of the sample block 12 when the multi-zone heater 156 is turned off and the sample block 12 loses heat. mainly through the control cooling channels. This is the situation during the control process carried out when the temperature of the sample block has deviated slightly above the desired target incubation temperature and the control system is trying to lower the temperature of the sample block to the temperature of user-specified incubation. Too fast a cooling rate in this situation could cause the desired incubation temperatures to be exceeded before the control system servo-feedback loop can respond even though a “controlled bypass” algorithm is used as described. will describe later. Since the block temperature servo feedback loop has a time constant to react to stimuli, it is desirable to control the amount of heat and cooling and the resulting rate of temperature change of the sample block such that overshoot is minimized. by not changing the temperature of the sample block at a rate greater than that which allows the system to respond to temperature errors.

In the preferred embodiment, the control cooling channels are 4mm in diameter, and the silicone rubber tube has an inner diameter of 1mm with a wall thickness of 1.5mm. This provides a control cooling rate of approximately 0.2 ° C per second when the block is at its high end of the operating range, i.e. near 100 ° C, and a control cooling rate of approximately 0, 1 ° C per second when sample block 12 is at a temperature at the low end of its operating range. The refrigeration control system 24 of Figure 1 causes the flow rate of refrigerant in the control cooling channels to be approximately 1/20 to 1/30 of the flow rate of liquid refrigerant through the rapid cooling channels, 100 to 107. The control cooling and rapid cooling channels are the same size, ie 4 mm in diameter, and extend completely through the sample block 12.

The control cooling channels are covered by inserting a rigid wire with a hook at the end of it through the control cooling channel and hooking it to a hole in the end of a silicone rubber tube, which has an outside diameter slightly larger than 4 mm. The cable hook is then placed through the hole in the silicone tube, and the silicone tube is pushed through the control cooling channel and cut flush with the end surface of the sample block 12.

Threaded holes 108-114 are used to secure refrigerant manifolds on each side of sample block 12. There is a threaded manifold on each end of the block. These two cooling manifolds are coupled to the cooling channels 26, 28, 30 and 32 of Figure 1, and they are fixed to the sample block 12 with a gasket material (not shown) interposed between the manifold and the metal of the block. of samples. These gaskets prevent the leakage of coolant and limit the thermal conductivity between the sample block 12 and the manifolds that represent a heat sink.

Any gasket material that serves the purposes stated above will suffice to practice the invention.

The positions of the control cooling and fast cooling channels in relation to the position of the slot 78 are best seen in the section of Figure 6. The positions of the control cooling and fast cooling channels in relation to the positions The sample wells are best seen in Figure 8. The control cooling and rapid cooling channels are generally interposed between the positions of the tips of the sample wells. Furthermore, Figure 8 reveals that rapid cooling and control cooling channels such as channels 106 and 97 cannot move much in the positive z direction without risking penetrating the wells of one or more sample wells. In the same way, the cooling channels cannot move much in the negative z direction without creating the possibility of penetrating the lower surface 116 of the sample block 12. For clarity, the positions of the control and rapid cooling channels are not shown in hidden lines in Figure 2 relative to the positions of the sample wells and other structures. However, there is either a control or rapid cooling channel between each column of sample wells.

Referring to Figure 2, the holes 118, 119, 120 and 121 are threaded and are used to attach the sample block 12 to the machinery used to machine the various holes and slots formed therein. In Figures 2, 4 and 5, the holes 124, 125, 126 and 127 are used to secure the sample block 12 to a fixing bracket shown in Figure 9 which will be described in detail below. Steel screws penetrating through this fixing bracket in threaded holes 124-127 provide mechanical fixation to sample block 12. These steel screws also represent heat sinks or heat sources that tend to add thermal mass to the sample block. sample block 12 and provide additional pathways to transfer thermal energy between sample block 12 and the surrounding environment. These fixing bolts and manifolds are two important factors in creating the need for guard bands that prevent heat energy transfer back and forth with these peripheral structures from affecting these sample temperatures.

ES 2 318 232 T3

Referring to figure 5, holes 128, 130 and 132 are mounting holes for the integrated circuit temperature detector (not shown) which is inserted into the sample block through hole 128 and secured there by screws. which fit into threaded holes 130 and 132. The degree of penetration of the object 128 and the relative position of the temperature detector with the slot 78 and the column adjacent to the sample wells are best seen in Figure 2.

Referring to Figure 2, objects 134 to 143 are mounting holes that are used to mount an overflow ring 147 (not shown). This overflow ring 147 is shown in Figure 19 which details the structure of the heated plate 14, the sliding cover 316, and the bolted clamp assembly 312. The purpose of this overflow ring is to prevent any liquid spilled from the overflow tubes. sample gets inside the instrument case where it could cause corrosion.

Referring to Figure 9, there is shown a sectional view of the fixing system and the configuration of the multi-zone heater 156 for the sample block 12. The sample block 12 is fixed by four screws of which the screw 146 is typical. These four screws pass through the vertical portions of a 148 steel fixing bracket. Two large springs 150 and 152 are compressed between the horizontal portion of the mounting bracket 148 and the steel pressure plate 154. The springs 150 and 152 are compressed sufficiently to provide approximately 21 kg / cm² (300 lb per square inch) force in the positive z direction acting to compress a film heater 156 against the bottom surface 116 of the sample block 12. This three-layer film heater structure is comprised of a multi-film heater zone 156, a silicone rubber sheet 158, and an epoxy resin foam layer 160. In the preferred embodiment the film heater 156 has three separately controllable zones. The purpose of the film heater 156 is to supply heat to the sample block 12 under the control of the CPU 20 of FIG. 1. The purpose of the silicone rubber sheet is to decrease the thermal conductivity from the film heater layer 156 to the lower structures. These lower structures serve as heat sinks and heat sources between which unwanted heat energy can be transferred to and from the sample block 12. The silicone rubber sheet 158 ​​has the additional function of compensating for the surface irregularities of the film heater 156 since the film heaters include nichrome wires and may not be perfectly flat.

The purpose of the steel plate 154 and the epoxy resin foam 160 is to transfer the force from the springs 150 and 152 to the silicone rubber sheet 158 ​​and the multi-zone film heater 156 and to compress the film heater against the surface. bottom 116 of the sample block as glued as possible. The epoxy resin foam should be rigid so as not to be crushed under the force of the springs but it should also be a good insulator and should have a low thermal mass, that is, it should be a non-dense structure. In one embodiment, resin 160 is manufactured under the ECKO foam trademark. In alternative embodiments, other structures can be substituted for the silicone rubber layer 158 and / or the epoxy resin foam layer 160. For example, a rigid panel structure such as that used in airplane construction could be placed between pressure plate 154 and film heater 156 with insulating layers between them. Any structure that is used for layers 158 and 160 should not absorb substantial amounts of heat from sample block 12 while the block is being heated and should not transfer substantial amounts of heat to sample block 12 when the block is being cooled. Perfect isolation of the block from the surrounding structures, however, is virtually impossible. Every effort should be made in the design of alternative structures that will be in contact with the sample block 12 to thermally isolate the sample block from its surroundings as much as possible to minimize the thermal mass of the block and allow for rapid changes in temperature of the sample. sample block and the sample mixes stored in it.

Precise control of the temperature of the sample block is achieved by the CPU 20 of Figure 1 by controlling the amount of heat applied to the sample block by the multi-zone film heater 156 of Figure 9. The film heater is driven by means of a modified form of pulse: width modulation. First, the 120-volt waveform from the power line is rectified to keep only half cycles of the same polarity. Then, portions of each half cycle is directed to the appropriate zones of the reed heater, with the CPU 20 being controlled by the percentage of each half cycle that is applied to each of the various zones of the reed heater.

Figure 10 illustrates one embodiment of a power control concept for reed heater 156. Figure 10 is a line-power voltage waveform diagram. Rectification takes place to remove the negative half cycle 162. Only the positive half cycles remain, of which the half cycle 164 is typical. The CPU 20 and its associated peripheral electronic circuitry then control the portion of each half cycle to be applied in accordance with the calculated power level for each zone based on the equations given below for each zone. That is, the dividing line 166 is moved forward or backward along the time axis to control the amount of power to the film heater based on a number of factors that are related in a special equation for each zone. The hatched area under the positive half cycle 164 represents the amount of power applied to the film heater 156 by the illustrated position of the split line 166. By moving the split line 166 to the right, more power is applied to the film heater and the sample block 12 is heated more. By moving the dividing line to the left along the time axis, the hatched area becomes smaller and less power is applied to the film heater. How the CPU 20 and its associated program and its peripheral circuits control the temperature of block 12 is described in detail below.

ES 2 318 232 T3

The amount of power supplied to the film heater is continuously variable from 0 to 600 watts. In alternative embodiments, the amount of power supplied to the film heater 156 can be controlled using other schemes such as computer control over the flow of current through it or the voltage applied to a direct current film heater or by the switching method. in the zero crossing described below.

In other embodiments, control of the heating of the sample block 12 can be accomplished by controlling the flow rate and / or the temperature of hot gases or hot liquid which is circulated in a controlled manner through the heating control channels which are form along sample block 12. Naturally in such alternative embodiments, the number of sample wells in the block would have to be reduced since there is no room for additional heating channels in the sample block 12 shown in Figures 2 to 8. Such alternative embodiments could still be compatible. with the 96-well microtiter plate format if, for example, one out of every two channels was removed to make room for the heating channel in the sample block. This would provide compatibility only in terms of the dimensions of such microtiter plates and not in terms of the simultaneous processing of 96 different samples. Care must be taken to preserve local balance and local symmetry in these alternative embodiments.

In the embodiment described herein, the maximum power that can be delivered to the block by means of the film heater is 1100 watts. This limitation comes from the thermal conductivity of the block / heater interface. It has been found experimentally that supplying more than about 1100 watts to the film heater 156 will frequently cause self-destruction of the device.

Typical power for heating or cooling when controlling the block temperature at or near the target incubation temperature is in the range of plus or minus 50 watts.

Referring to Figure 11, a plot of temperature versus time is shown for a typical PCR protocol. Large decreases in block temperature are accomplished by controlled controlled circulation of cooled liquid refrigerant through rapid cooling channels while monitoring the temperature of the sample block by temperature detector 21 of Figure 1. Typically these rapid decreases in temperature take place during the subsequent ramp from denaturation incubation 170 toward hybridization incubation temperature 172. Typically, the user must specify the protocol defining the temperatures and times in one way or another to describe to the CPU 20 the positions on the temperature / time plane of the checkpoints symbolized by the circled intersections between the ramp stages and the stages. incubation. Generally, the incubation steps are marked with the reference numbers 170, 172 and 174 and the ramps are marked with the reference numbers 176, 178 and 180. Generally the incubation intervals are performed at a single temperature, but in alternative embodiments it may be stepped or continuously varied at different temperatures within a temperature range that is acceptable to perform the particular portion of the PCR cycle involved. That is, denaturation incubation 170 need not be carried out at a temperature as shown in FIG. 11, but can be carried out at any of a variety of different temperatures within the range of temperatures acceptable for denaturation. In some embodiments, the user can specify the length of ramp segments 176, 178, and 180. In other embodiments, the user can only specify the temperature (s) and duration of each incubation interval, and the instrument will then change the temperature of the sample block as quickly as possible between incubation temperatures from the end of an incubation to the beginning. of other. In the preferred embodiment, the user may also have incubation temperatures and / or times that are different for each cycle or that are automatically increased with each cycle.

The mean power of flash cooling during a transition from denaturation incubation at 95 ° C to hybridization incubation at 35 ° C is typically greater than one kilowatt. This produces a temperature change in the sample block of approximately 4-6 ° C per second when the temperature of the block is at the upper end of the operating range, and approximately 2 ° C per second when the temperature of the block is the lower end. of the operating range. It is generally desirable to have as high a rate of cooling as possible for rapid cooling.

Because a lot of heat is removed from the sample block during flash cooling, temperature gradients can occur across the sample block from one end of the quick chill channel to the other. To prevent this and minimize these types of temperature gradients, the rapid cooling channels are interspersed with their directions. That is, in Figure 3, the direction of the cooling flow through the rapid cooling channels 100, 102, 104, and 106 is towards the page as symbolized by the X's inside the hole of these cooling channels. fast. The flow of the liquid in the rapid cooling in the interleaved rapid cooling channels 101, 103, 105, and 107 is directed away from the page as symbolized by the point in the center of the hole in these rapid cooling channels. This interleaving plus the high flow rate through the rapid cooling channels minimizes any temperature gradients that might otherwise occur using non-interleaved flow patterns or lower flow rates because the distances from hot spots to cold spots have been made shorter. . A lower flow rate would result in most or all of the heat being taken from the block in the first two centimeters or so of the path which means that the inlet side to the block will be at a lower temperature than the outlet side of the block. A high flow rate minimizes the temperature gradient along the channel. Sandwiched means that the hot end of the channels flowing in one direction are interposed between the cold ends than the channels in which the flow is in the opposite direction. This is a distance less than the length of the channel. Thus, the

ES 2 318 232 T3 temperature gradients are reduced because in the distances that the heat must travel to eliminate the temperature gradient is reduced. This causes any temperature gradients that form due to the chill channels to be quickly eliminated before they have time to differentially heat some samples and not others. Without interleaving, one side of the sample block would be approximately 1 ° C higher than the other side. Interleaving produces dissipation of any temperature gradients that may be caused in less than approximately 15 seconds.

To accurately estimate the amount of heat added or removed from the block, the CPU 20 measures the temperature of the block using the temperature sensor 21 of figure 1 and measures the temperature of the coolant by means of the temperature sensor 61 in figure 46 attached. to bus 54 in figure 1. The ambient air temperature is also measured by means of the temperature detector 56 in FIG. 1, and the power line voltage is also measured, which controls the power applied to the film heaters on the bus 52. The thermal conductivity from the sample block to the environment and from the sample block to the coolant are known to the CPU 20 as a result of measurements made during the initialization process to adjust the system control parameters.

For good temperature uniformity in the sample population, the block, at constant temperature, cannot have a net heat input or output. However, temperature gradients may occur within the sample block arising from local heat flows from hot spots to cold spots giving a net zero heat transfer relative to the edges of the block. For example, a piece of material that is heated at one end and cooled at the other is at a constant mean temperature if the net heat flux in the block is zero. However, in this situation a significant temperature inequality, ie a temperature gradient, can be established within the piece due to the flow of heat from the hot edge to the cold edge. When the cooling heating of the edges of the block is finished, the heat flow from the hot edge to the cold edge finally dissipates this temperature gradient and the block reaches a uniform temperature across it which is the average between the hot and cold temperature. the cold temperature at the beginning of the heat flow.

If a material of sectional area A and length L has a uniform thermal conductivity K, and the material is kept at a constant temperature due to the input from the heat source Q_in fits outlet on heat sink Q_out, the stable temperature profile that results from the heat flow is:

<img file="ES2318232T3_D0001.tif" />

Where,

Delta T = temperature gradient

L =

A =

K = the length of the thermal path the area of ​​the thermal path the thermal conductivity along the path

In general, within any material of uniform thermal conductivity, the temperature gradient will be set in proportion to the heat flux per unit area. Heat flow and temperature inequality are thus closely linked.

In practical terms, it is not possible to control the temperature of the sample block without some incoming or outgoing heat flow. Control cooling requires some incoming heat flow to the strip heaters to balance the heat removed by the coolant flowing through the control cooling channels and keep the block temperature at a stable value. The key to a uniform sample block temperature under these conditions is a geometry that has "local equilibrium" and "local symmetry" of heat sources and heat sinks both statically and dynamically, and which is arranged so that any heat flow from hot spots to cold spots occurs only over short distances.

In short, the concept of "local static equilibrium" means that in a constant temperature block where the total heat input equals the total heat output, the heat sources and heat sinks are arranged such that within a local region Defined, all heat sources are fully balanced with heat sinks in terms of heat entering and heat leaving the block. Therefore, each local region, if isolated, would be kept at a constant temperature.

The concept of "local static symmetry" means that, within a local region and for a constant temperature, the center of mass of the heat sources coincides with the center of mass of the heat sinks. If this is not the case, within each local region there may exist across each local region a temperature gradient that

ES 2 318 232 T3 can add to a temperature gradient in an adjacent local region thereby causing a gradient across the sample block that is as large as twice the size of a simple local region due to the absence of even local symmetry. although there is a local balance within each local region. The concepts of local equilibrium and local symmetry are important in achieving a static temperature equilibrium where the temperature of the sample block is maintained at a constant level during, for example, an incubation interval.

For the dynamic case in which rapid temperature changes take place in the sample block, the thermal mass, or heat capacity of each local region becomes important. This is because the amount of heat that must flow to each local region to change its temperature is proportional to the thermal mass of that region.

Therefore, the concept of static local equilibrium can be extended to the dynamic case by requiring that if a local region includes x percent of the total dynamic heat sources and sinks, it must also include x percent of the thermal mass for there to be a "Local dynamic balance". In the same way, "local dynamic symmetry" requires that the center of mass of the heat capacity coincide with the center of mass of the dynamic heat sources and sinks. What this means in simple terms is that the thermal mass of the sample block is the metal itself, and that the machining of the sample block must be symmetrical and balanced such that the total mass of metal within each local zone is the same. Additionally, the center of mass of the metal in each local zone should coincide with the center of mass of the dynamic heat sources and sinks. Thus, the center of mass of the multi-zone heater 156, that is, its geometric center, and the geometric center of the control and fast cooling channels must coincide. From the study of Figures 2-9, it can be deduced from the comments below that both dynamic and static local symmetry and equilibrium exist in sample block 12.

Figure 12 illustrates two contiguous local regions for the design of sample block 12 in accordance with the teachings of the invention. In Figure 12, the boundaries of the two local regions, 200 and 202, are marked by the dashed lines 204, 206, and 208. Figure 12 shows that each local region that is not in the guard band is composed of: two columns of sample wells; a portion of the film heater 156 becoming 1/8 of the total area of ​​the heater; a rapid cooling channel such as rapid cooling channels 210 and 212; and a control cooling channel. To preserve local symmetry, each local region focuses on its fast cooling channel and has half a control cooling channel at each boundary. For example, region 200 is centered on rapid cooling channel 210 and control cooling channels 214 and 216 are divided by the boundaries of local region 204 and 206, respectively. Thus the center of mass of the rapid cooling channel (the center of the same), coincides (horizontally) with the center of mass of the control cooling channels (the center of the local region) and with the center of mass of the portion of the film heater attached to each local region. There will be a local static balance in each local region as CPU 20 drives film heater 156 to introduce an amount of heat energy that is equal to the amount of heat energy that is being extracted by the fast cooling and control cooling channels. There is local dynamic balance for each local region because each local region in the central part of the block where the 96 sample mixtures reside contains approximately 1/8 of the total thermal mass of the entire sample block, it contains 1/8 of the total number of fast cooling channels and contains 1/8 of the total number of control cooling channels. There is local dynamic symmetry for each local region because the center of mass of each local region coincides horizontally with: the center of the portion your film heater in front of the local region; the center of the rapid cooling channel; and the center of mass of the two halves of the control cooling channels.

Thanks to these physical properties characterized as local static and dynamic balance and symmetry, the sample block heats and cools all samples in the population much more uniformly than prior art thermal cycling performers.

Referring to Figure 2, the plan view of the boundaries of the local regions is illustrated by the dashed lines 217 to 225. Inspection of Figure 2 reveals that the central region of the 96 sample wells is divided into six local regions adjacent boundaries marked by boundaries 218 to 224. In addition, two local protection regions are added at each edge. The local edge regions (local regions are also sometimes called local cones here) that have the most negative x-coordinate are marked by the boundary lines 217 and 218. The local region of the edge having the most positive x-coordinate is marked by boundary lines 224 and 225. Note that the local regions of the edge do not contain columns of sample wells but instead contain slot 78 simulating a column of wells. The depth and width of the groove 78 is designed to extract the same metallic mass as a column of wells and hence the local dynamic symmetry is preserved in some way. The local edge zones are therefore different in thermal mass (they also have additional thermal mass thanks to external connections such as manifolds and clamping bolts) than the six local zones in the central part of the sample block. This difference is taken into account for the heating of the local edge zones or protection bands with zones that can be controlled separately from said multi-zone heater so that more energy is introduced into the protection band than in the central zone of the block.

The local regions of each edge of the block approximate, but do not exactly match the thermal properties of the six local regions located in the center. The local edge regions are called "guard band" regions because they complete a guard band that runs around the periphery of sample block 12. The purpose of this guard band is to provide some thermal insulation to the central portion of the sample block containing the 96 sample wells from inherently uncontrolled heat sources and sinks.

ES 2 318 232 T3 made on the mechanical connections to the block by objects such as support bolts, collectors, overflow rings and other devices that must be mechanically fixed to the sample block 12. For example in figure 2, the edge of the Surfaces 228 and 230 of the sample block have attached plastic manifolds that carry coolant to and from the rapid cooling and control passages. The guard band along the edges 228 and 230 consists of portions of the groove 78 that are parallel to and close to the edges 228 and 230. The depth of the groove 78 is such that the bottom of the groove approaches the perimeters of the control and rapid cooling channels as much as possible without actually intersecting them. The width of the groove 78 along with its depth is such that the volume of metal removed by the groove between points 82 and 232 of Figure 2 approximately equals the volume of metal removed by the adjacent row of sample wells beginning with the well. sample 234 and ending with sample well 83. Also, the slot 78 around the perimeter of the block is located approximately where an additional row of wells would be if the periodic pattern of sample wells were to extend one row or column of wells in each direction.

Along the edges 250 and 252 where the clamp connections to the sample block are made, the local regions of the guard band contain, in addition to a portion of the slot 78, the full length of various cooling channels. Referring to Figure 3, these include: 1/2 control cooling channel (e.g. 92) merging with 1/2 control cooling channel from the adjacent local region to form a complete cooling channel of control; a fast cooling channel (eg 100); and a complete control cooling channel (eg 91). For the local edge region at edge 250, these cooling channels are 107, 198, and 99.

All control cooling channels in the guard strips are slightly offset inward from the edge of the block. The reason these complete control cooling channels are used is because "half" cooling channel is not possible to build. Since the control cooling channels require the lining with thick rubber walls, it would be difficult to keep a hole through the "half" lining of the control cooling channel reliably open. This asymmetry in the local edge regions causes a small excess of heat loss to the coolant from the local regions of the edge guard band, but is far enough away from the central region of the sample block containing the sample wells as so that its contribution to inequalities in sample temperatures is small. Also, because the temperature effects of this small asymmetry are predictable, the effect can be further minimized by using a separately controllable zone of the multi-zone heater system under each guard band.

Referring to FIG. 13, three separately controllable zones are shown within the film heater layer 156 of FIG. 9. These separately controllable zones include edge heater zones that are located under the guard strips at the exposed edges. of sample block 12 that are attached to fixture 148. There are also separately controllable manifold heater zones located under edge guard bands 228 and 230 that are attached to the cooling manifolds. Finally, there is an area of ​​the central heater that is under the sample wells. The power applied to each of these zones is controlled separately by the CPU 20 and the control program.

Film heater 156 is comprised of a pattern of electrical conductors formed by etching a thin sheet of metallic alloy such as Inconel *. The selected metal alloy should have high electrical resistance and good heat resistance. The pattern of conductors thus etched is glued between thin sheets of an electrically insulating polymeric material such as Kapton *. Whatever the material used to insulate the heating element by electrical resistance, the material must be resistant to high temperatures, have a high dielectric coefficient and good mechanical stability.

The central zone 254 of the film heater has approximately the same dimensions as the central portion of the sample block within the guard strips. Central region 254 delivers a uniform power density to the sample well area.

Edge heater regions 256 and 258 are almost as wide as the edge guard bands but are not as long.

The manifold heating regions 260 and 262 are under edge guard bands 228 and 230 of FIG. 2.

The manifold heating zones 260 and 262 are electrically connected together to form a separately controllable heating zone. Also, edge heater sections 256 and 258 are electrically connected together to form a second separately controllable heating zone. The third separately controllable heater zone is the center section 254. Each of these three separately controllable heater zones has separate electrical terminals, and each zone is controlled by a separate control algorithm that can be run on separate microprocessors or on a shared CPU as is done in the preferred embodiment.

Edge heater zones 256 and 258 are activated to compensate for heat losses from the fixture brackets. This heat loss is proportional to the temperature difference between the sample block 12 and the air.

ES 2 318 232 T3 surrounding environment. Edge heater zones 256 and 258 also compensate for excess heat loss from the sample block to the complete control cooling channels at each edge of the block. This heat loss is proportional to the difference in temperature between the sample block 12 and the refrigerant circulating through these control cooling channels.

Manifold heater sections 260 and 262 are also activated to compensate for heat loss to plastic cooling manifolds 266 and 268 of FIG. 13 that are attached to the edges of sample block 12. Power to manifold heater sections 260 and 262 compensates for heat losses that are fundamentally proportional to the temperature difference between the sample block and the coolant and, to a lesser degree, between the sample block and ambient air .

For practical reasons, it is not possible to match the thermal mass of the local regions of the guard band with the thermal masses of the local regions that include the sample wells arranged on the central section of the heater 254. For example, plastic cooling manifolds 266 and 268 not only conduct heat outside of the guard band, but also add a certain amount of thermal mass to the local regions of the guard band to which they are attached. The result of this is that during rapid changes in block temperature, the rates of rise and fall of the temperature of the guard band do not exactly match that of the local regions of the sample wells. This generates a dynamic temperature gradient between the guard bands and the sample wells, and if allowed to become large, it could remain for a longer time than is tolerable. This temperature gradient effect is roughly proportional to the rate of change in block temperature and is minimized by adding or removing heat from each local zone of the buffer at a rate that is proportional to the rate of change in temperature of the block.

The proportionality coefficients for the guard band zone heaters are relatively stable system design properties, and are determined by engineering measurements on the prototypes. The values ​​for these proportionality coefficients are given below in relation to the definitions of the terms in equations (3) to (5). These equations define the amounts of power to be applied to the manifold heater zone, edge heater zone, and center zone, respectively in an alternative embodiment. The equations used in the preferred embodiment will be given later in the program description (equations (46) - (48), distributed power per area).

(3) P_m = A_m P + Kmi (TblQ - Tamb) + Km2 (TblQ - TrEFr) + Km3 (dBLQ / dt) where,

P_m = the power supplied to manifold heater zones 260 and 262.

TO_m = the area of ​​the manifold heater zone.

P = power required for the block temperature to remain at or move to the desired temperature at any particular time in a PCR thermal cycling protocol.

K_M1 = a constant of proportionality determined experimentally to compensate for the excess heat loss to the environment through the collectors, equal to 0 watts / degree Kelvin.

K_m2 = an experimentally determined constant of proportionality to compensate for excess heat loss to the refrigerant, equal to 0.4 watts / degree Kelvin.

K_m3 = a constant of proportionality determined experimentally to give an additional power to compensate for the additional thermal mass of the protection bands in the collectors produced by the fixing of the plastic collectors, etc., equal to 66.6 watt-seconds / degree Kelvin.

T_BLQ = the temperature of sample block 12.

T_AMB = the ambient air temperature.

Trefr = the temperature of the coolant.

dt_BLQ/ dt = the change in temperature of the sample block per unit time.

ES 2 318 232 T3 (4) Pe - Ae Ρ + Kei (Tblq - Tamb) + Ke2 (Tblq - Trefr) + Ke3 (dÍBLQ / dt) where,

P_AND = the power to be applied to the edge heater zones.

TO_and = the area of ​​the edge heater zone.

K_E1 = a constant of proportionality determined experimentally to compensate for excess heat loss to the environment through the collectors, equal to 0.5 watts / degree Kelvin.

K_E2 = an experimentally determined constant of proportionality to compensate for excess heat loss to the refrigerant, equal to 0.15 watts / degree Kelvin.

K_E3 = an experimentally determined constant of proportionality to give an additional power to compensate for the additional thermal mass of the exposed protection bands produced by the fixing to the sample block 12 of fixing and support bolts, the temperature detector, etc., equal to 15.4 watt seconds / degree Kelvin.

<img file="ES2318232T3_D0002.tif" />

where,

P_C = the power to be applied to the central zone 254 of the multi-zone heater.

TO_C = the area of ​​the central zone 254.

In each of equations (3) to (5), the power term, P, is a variable that is calculated by the portion of the control algorithm run by CPU 20 of Figure 1 that reads the user-defined set points and determines what to do next to make the sample block temperature stay at or reaches the appropriate temperature to implement the PCR temperature protocol defined by the time and temperature set points stored in memory by the user. The way in which the set points are read and the power density is calculated will be described in more detail below.

The control algorithm executed by the CPU 20 of figure 1 detects the temperature of the sample block through the temperature detector 21 of figure 1 and figure 9 and the S bus₂ from Figure 1. This temperature is differentiated to obtain the rate of change of the temperature of the sample block 12. The CPU then measures the ambient air temperature through the temperature detector 56 of Figure 1 and measures the temperature of the coolant through the temperature sensor 61 in the refrigeration control system 24 shown in FIG. 46. The CPU 20 then calculates the power factor corresponding to the particular segment of the PCR protocol that is being implemented and performs three calculations according to equations (3), (4) and (5) by inputting all the measured temperatures, the constants proportionality (which are stored in non-volatile memory), the power factor P for that particular integration of the control program and the areas of the various heater zones (which are stored in non-volatile memory). Power factor is the total power required to change the block temperature from its current level to the user-specified temperature level through the set points. Further details on the calculations performed by the CPU to control heating and cooling are given in the description of the "PID task" control program.

After calculating the power required to be applied to each of the three zones of heater 156, another calculation is made regarding the proportion of each half cycle of the input voltage to be applied to each zone in some embodiments. In the preferred embodiment described below, the calculation mode is how many half cycles of the total number of half cycles that occur during a 200 millisecond sample period are to be applied to each zone. This process is described below in connection with the discussion of Figures 47A and 47B (hereinafter referred to as Figure 47) and the "PID task" of the control program. In an alternative embodiment symbolized by FIG. 10, the calculator finds, for each zone, the position of the division line 166 of FIG. 10. After the calculation is performed, the appropriate control signals are generated to cause the power sources for the multi-zone heater 156 to perform the appropriate switches to cause the amount of power calculated for each zone to be applied thereto.

In alternative embodiments, the multi-zone heater can be implemented using a single film heater that delivers a uniform power density to the entire sample block, plus one or two additional film heaters with only one zone each for the guard bands. These additional heaters are

ES 2 318 232 T3 superimposed on top of the single film heater that covers the entire sample block. In such an embodiment, only the necessary power is delivered to the additional heaters to cover the losses of the guard band.

The power factor that is calculated by the CPU 20 in equations (3) through (5) for various points of the PCR temperature protocol based on the user specified change time set points. However, a limitation is imposed based on the maximum power delivery capacity of the aforementioned zone heater.

The constants of proportionality in equations (3) to (5) must be set appropriately to adequately compensate for excess heat losses in the shield band for good temperature uniformity.

Referring to Figure 17, it shows a graph of the differences between the calculated sample temperatures for a variety of different samples in response to a step change in block temperature that raises the temperature of the sample block towards the temperature. Target for denaturation incubation of about 94 ° C from substantially lower temperature. Figure 17 illustrates the liquid temperatures of the samples calculated when the lime multi-zone heater 156 is properly managed using the constants of proportionality given above in the definitions of the terms for equations (3) through (5). The various wells that were used to produce the graph of Figure 17 are indicated therein by a simple number and letter combination. The 8 x 12 well array shown in Figure 2 is coded by columns with letters and rows with numbers. Thus, for example, sample well 90 is also designated as sample well A12, while sample well 89 is also designated as sample well B12. In the same way, sample well 68 is also designated sample well D6, and so on. Note that the well temperatures are asymptotically at temperatures that are within approximately 0.5 ° C of each other, due to the general thermal design described herein to eliminate temperature gradients.

The above description illustrates how the temperature of the sample block can be controlled to be uniform and rapidly changeable. However, in the PCR process, it is the temperature of the sample reaction mix and not the temperature of the block that has to be programmed. In the preferred embodiment in accordance with the teachings of the invention, the user specifies a sequence of target temperatures for the liquid sample itself and specifies incubation times for the liquid sample at each of these target temperatures for each step in the process. PCR. CPU 20 then manages the temperature of the sample block to have the sample reaction mixes at the specified target incubation temperatures and maintain the sample mixes at these target temperatures for the specified incubation times. The user interface code executed by CPU 20 shows on the display of terminal 16, at all stages of this process, the current temperature of the calculated liquid sample.

The difficulty in showing the actual temperature measurement of the sample is that measuring the actual temperature of the reaction mixture requires the insertion of a temperature measurement probe into it. The thermal mass of the probe can significantly alter the temperature of any well it is placed in since the sample reaction mix in any particular well is often only 100 microliters in volume. Thus, the mere insertion of a temperature probe into a reaction mixture can cause a temperature gradient to exist between that reaction mixture and neighboring mixtures. Since the extra thermal mass of the temperature detector would cause the reaction mixture to be immersed in lag behind in temperature relative to the temperatures in reaction mixtures in other wells that have lower thermal mass, errors in amplification simply by trying to measure temperature.

Consequently, the instrument described here calculates the sample temperature from known factors such as the block temperature history and the system thermal time constant and displays this sample temperature on the screen. It has been found experimentally for the system described herein that if the mixing tubes are pressed down against the sample wells with at least a minimum limiting force F, then for the size and shape of the sample tubes used in the preferred embodiment and sample volumes of approximately 100 microliters, thermally activated convection occurs within the sample reaction mixture and the system acts thermally as a linear system, with a single time constant. Experiments have shown that each sample tube must be pushed down with approximately 50 grams of force for good well-to-liquid wall thermal conductivity from well to well. The heated plate design described below is designed to push down each sample tube with about 100 grams of force. This minimal force, symbolized by the force vector F in Figure 15, is necessary to ensure that regardless of slight differences in external dimensions between various sample tubes and various sample wells in the sample block, they will all be pushed down with enough force to ensure tight and aligned fit of each tube ensuring uniform thermal conductivity. Any design that has some sample tubes with a loose fit in their corresponding sample wells and some tubes with tight fittings will not be able to achieve equal temperature control for all tubes due to non-uniform thermal conductivity. An insufficient force level F results in a temperature response of the liquid sample to a step change in block temperature as shown at 286 of Figure 14. A suitable force level F results in the temperature response shown at 282.

The result obtained by the apparatus constructed in accordance with the teachings of the invention is that the temperature of each sample mixture behaves as if the sample were physically well mixed during transitions.

ES 2 318 232 T3 at the new temperatures. In fact, due to the convection currents produced in each sample mixture, the sample reaction mixture in each sample tube is well mixed.

The surprising result is that the thermal behavior of the complete system is like that of an RC electrical circuit with a simple time constant of 9 seconds that is around 1.44 times the half-life of the decrease in the difference between the block temperature and the temperature of the sample. A GeneAmp * sample tube filled with 50 milliliters of sample has a time constant of about 23 seconds. In other words, during an upward change in the temperature of the sample block, the temperature of the reaction mixture acts as the voltage rise of a capacitor C in an RC electrical circuit as shown in Figure 16D in response to a change in step in the output voltage of the voltage source V.

To illustrate these concepts, we refer to Figure 14 which shows the different temperature responses of the liquid sample to a step change in block temperature and to Figure 15 which shows a section through a sample well / combination of sample tube. It has been found experimentally that when the volume of the liquid sample 276 is approximately 100 microliters and the dimensions of the tube are such that the meniscus 278 is located below the upper surface 280 of the sample block 12, and the force F pushing the sample tube to the sample well is at least 30 grams, the thermal time constant τ varies by about one second for every 0.025 mm change in wall thickness for the frusto-conical sample tube. The thin-walled sample tubes described herein have been found to have thermal time constants of about 5 to about 14 seconds when they contain 20 to 100 microliters of sample. Thicker tube walls would result in higher time constants and a longer delay between a change in the temperature of the sample block and the resulting change in the temperature of the liquid sample.

Mathematically, the expression for the thermal response of the temperature of the liquid sample to a change in the temperature of the sample block is:

(®) T_deadstra ⁼ ΔΤ (1 - β ^ϋτ) where,

T_deadstra = the temperature of the liquid sample

AT = the temperature difference between the temperature of the sample block 12 and the temperature of the liquid sample t = elapsed time _T = thermal time constant of the system, or the heat capacity of the sample divided by the thermal conductivity between the wall of the sample well and the liquid sample

In Figure 14, curve 282 represents the exponential temperature response to a theoretical step change in the temperature of the sample block when the force F pushing down the sample tube is high enough. The step change in the temperature of the sample block is indicated as function 284, with a rapid rise in temperature beginning at time Ti. Note how the temperature of the liquid sample increases exponentially in response to the change in step and asymptotically approaches the final temperature of the sample block. As briefly mentioned earlier, curve 286 represents the thermal response when the downward seating force F in Figure 15 is sufficient to produce a tight, aligned fit between the cone of the sample tube and the wall 290 of the sample well. . Generally, the thermal response from curve 286 will result if the force F is less than 30 grams. Note that although Figure 15 indicates a small layer of air between the cone of the sample tube and the wall of the sample well for clarity, this is the exact opposite of the desired situation since air is a good insulator and it would substantially increase the thermal time constant of the system.

The thermal time constant τ is analogous to the RC time constant of an RC series circuit where R corresponds to the thermal resistance between the wall of the sample well and the sample liquid and C is the heat capacity of the sample liquid. sample. Thermal resistance is equal to the inverse of thermal conductivity which is expressed in units of watt seconds per degree Kelvin.

Because of the convection currents 292 noted in the sample liquid of Figure 15, all locations in the sample liquid reaction mixture are very close to the same temperature; the heat flux between the block and the sample is very closely proportional to the temperature difference between the sample block and the sample reaction mixture. The constant of proportionality is the thermal conductivity between the wall of the sample well in sample block 12 and the reaction mixture. For different sample volumes or different tubes, i.e. different wall thicknesses or materials, the thermal time constant will be different. In such a case, the user can, as part of their PCR protocol specification, enter the sample volume or the

ES 2 318 232 T3 tube type and the machine will automatically search for the correct thermal time constant to use in calculating the sample temperature. In some embodiments, the user can enter the real-time constant, and the machine will use it to calculate the sample temperature.

To keep the thermal time constant as small as possible, the conical walls of the sample tubes should be as thin as possible. In the preferred embodiment, these conical walls are 0.23 mm thick while the walls of the cylindrical part of the sample tube are 0.76 mm thick. The conical shape of the sample tube provides a relatively large metal contact surface of the sample well wall relative to the volume of the sample mixture.

The molding of the sample tubes is performed using a "cold run" system and a four-cavity mold in which four sample tubes are molded with each injection. Molten plastic is injected into the tip of the tube cone so that any remaining plastic will project into cavity 291 between the tip of the sample tube and the tip of the sample well. This prevents any debris from interfering with the aligned fit between the tube and the well. A maximum limit of 0.76 mm is set for the size of any plastic debris.

In various embodiments, three different grades of polypropylene can be used each with their different advantages. The preferred polypropylene is Himont PD701 because it is autoclavable. However, this plastic is difficult to mold due to its low melt index. This plastic has a melt index of 35 and a molecular density of 9. PD701 tends to stain and create somewhat uneven quality parts but would work better if it were injected into the thin wall part of the mold rather than the tip of the tapered section as is normally done. Generally, it is desirable that it have a high melt index for easy molding but also a high molecular density to maintain good rigidity and prevent cracking or breakage under the heat stress of the autoclaving process at 127 ° C. Another plastic, American Hoescht's PPW1780 has a melt index of 75 and a molecular density of 9 and is autoclavable. Another plastic that can be used in some embodiments is Himont 444. This plastic is not autoclavable and needs to be sterilized otherwise.

In alternative embodiments, the tubes can be molded using a "hot runner" or "hot nozzle" system where the temperature of the molten plastic is controlled until it exits the mold. Also, in some embodiments, multiple outlets can be used. However, none of these techniques have been experimentally proven at the time of this writing to be better than the currently used "cold run" system.

The fact that the system acts thermally as a simple time constant RC circuit is an important result, because it means that if the thermal conductivity from the sample block to the sample reaction mixture is known and uniform, the thermal response of the Sample mixes will be known and uniform. Since the heat capacity of the sample reaction mixture is known and constant, the temperature of the sample reaction mixture can be accurately calculated using only the history of the block temperature measurements over time. This eliminates the need to measure the temperature of the sample thereby eliminating the errors and mechanical difficulty of placing a probe with a non-negligible thermal mass in a sample well to measure the temperature of the sample directly and thereby change the thermal mass of the sample. the sample in the tested well.

The algorithm that performs this calculation imitates the thermal behavior of the system in the manner of a series RC electric circuit with a simple time constant. That model uses the ratio of the heat capacity of the liquid sample divided by the thermal conductivity from the sample block to the sample reaction mixture. The heat capacity of the sample reaction mixture is equal to the specific heat of the liquid multiplied by the mass of the liquid. The thermal resistance is equal to one divided by the thermal conductivity from the sample block to the liquid reaction mixture through the walls of the sample tube. When this ratio of heat capacity divided by thermal conductivity is expressed in consistent units, it has the dimension of time. For a fixed sample volume and a fixed sample composition both of which are the same in each sample well and a fixed thermal conductivity, the relationship is also a constant for each sample well, and is called the thermal time constant. of the system. It is the time needed by the temperature of the sample to reach 36.8% of the temperature of the block after a sudden change in the temperature of the block.

There is a mathematical theorem used in the analysis of electronic circuits that maintains that it is possible to calculate the output response of a filter or other linear system if the response of the system to an impulse is known. This impulse response is also known as the transfer function. In the case of an RC series circuit, the impulse response is an exponential function as shown in Figure 16A. The impulse stimulus that results in the response of FIG. 16A is as shown in FIG. 16B. The aforementioned mathematical theorem maintains that the output response of such a linear system can be determined by calculating the convolution of the input signal and a weighting function where the weighting function is the impulse response of the system reversed in time. Convolution is also known as a weighted moving average even though a convolution is a computational concept with infinitely small intervals whereas a weighted moving average has discrete intervals, that is, multiple samples. The impulse response of the RC series circuit shown in Figure 16D is such that when the voltage of the generator voltage V suddenly rises and falls with a voltage spike as shown in Figure 16B, the voltage of the capacitor C increases rapidly. to a peak at 294 of Figure 16A which is equal to the peak voltage of the impulse shown in Figure 16B and then decays exponentially towards its

ES 2 318 232 T3 steady state of voltage V₁. The resulting weighting function is the impulse response of Figure 16A reversed in time as shown in Figure 16C at 385.

Superimposed on FIG. 16C is a hypothetical curve 387 illustrating a typical temperature history for the temperature of sample block 12 for an approximate step temperature change. Also superimposed on FIG. 16C are the times of five labeled temperature sample periods. In accordance with the teachings of the invention, the temperature of the sample is calculated by multiplying the temperature of each of these times T₁ to T₅ by the value of the weighting function at that particular time and then adding all these products and dividing them by 5. The fact that the thermal system acts as a simple time constant linear circuit is a surprising result based on the complexities of the heat transfer considerations in this complicated thermal system.

In one embodiment, the sample temperature calculation is adjusted with a short delay to account for the delay in transport caused by different lengths of thermal path for the block temperature detector and for the liquid sample. The calculated sample temperature is displayed for user information on screen 16 indicated in figure 1.

Figure 17 shows the results of the temperature response in six different wells spread across the 96-well sample block to a step change in the sample block temperature from a relatively low temperature in the temperature range of the sample. hybridization / extension at the relatively high temperature of about 94 ° C used for denaturation. The graph in Figure 17 shows a good agreement between the predicted exponential rise in the sample temperature if the system were perfectly analogous to the series RC circuit shown in Figure 16D, and also shows excellent uniformity of temperature response in that the temperatures of the six sample wells used for this study asymptotically approximate temperatures very close to each other and in a denaturation temperature "tolerance" band that has an amplitude of about 0.5 ° C.

In one embodiment, the 10 most recent block temperature samples are used to run a weighted average, but in other embodiments a different number of historical temperature samples may be used. The good agreement with the theoretically predicted results comes from the fact that the thermal convection currents produce a good mixture of the sample liquids, thereby making the system act linearly.

Uniformity between sample temperatures in various sample wells spread across the 96-well array is the result of local equilibrium and local static and dynamic symmetry in the sample block structure as well as all other design factors. detailed here. Note however that during rapid temperature changes all sample wells will have temperatures differing 0.5 ° C from each other only if the user has carefully filled each sample well with the same sample mass of the liquid sample. Inequalities in mass in different wells do not produce unequal temperatures in a steady state, with unchanging conditions, only during rapid changes. The mass of the liquid sample in each well is the dominant factor in determining the heat capacity of each sample and is therefore the dominant factor in the thermal time constant for that particular sample well.

Note that the ability to cause the liquid sample in all sample wells to rise and fall in temperature in unison and stabilize at target temperatures very close to each other, that is, in tolerance bands that have only a 0.5 amplitude. ° C, also depends on the force F in figure 15. This force must exceed a minimum limiting force before the thermal time constants of all sample wells filled with similar masses of the liquid sample have the same time constant. This minimum level of force has been experimentally determined to be 30 grams for the sample tube and sample well configuration described herein. For higher levels of precision, the minimum force level F in Figure 15 should be set to at least 50 grams and preferably 100 grams to have an additional margin of safety as stated above.

The importance of thermal uniformity in the temperature of the sample well can be appreciated by referring to Figure 18. This figure shows the relationship between the amount of DNA generated in a PCR cycle and the actual temperature during the denaturation interval of an example of a amplification of a certain segment of DNA. The slope of the 298 function between temperatures 93 and 95 degrees centigrade is approximately 8% per degree centigrade for this particular case of DNA and primers. Figure 18 shows the general shape of the curve in relation to the amount of DNA generated by amplification, but the details of the shape of the curve will vary with each different case of primers and target DNA. Temperatures for denaturation above 97 degrees centigrade are generally too hot and result in decreased amplification with increasing denaturation temperature. Temperatures between 95 and 97 degrees Celsius are generally just right.

Figure 18 illustrates that any sample well that contains this particular primer and target DNA combination and that is set at a denaturation temperature of approximately 93 ° C will commonly have 6% less DNA generated during a typical PCR protocol. than wells denatured at 94 ° C. In the same way, liquid samples from this mix that stabilize their denaturation temperatures at 95 ° C will commonly have 8% more DNA generated here than is generated in sample wells that stabilize at

ES 2 318 232 T3 denaturation temperatures of 94 ° C. Because all curves of this nature have the same general shape, it is important to have a uniformity in the temperature of the sample.

The sample temperatures calculated as described above are used by the control algorithm to control the heaters and flow through the blast chill channels and determine how long the samples have been held at various target temperatures. The control algorithm uses these times for comparison with the desired incubation period times as entered by the user. When the times match, the control algorithm takes appropriate action to heat or cool the sample block toward the user-defined target temperature for the next incubation.

When the calculated sample temperature is within one degree centigrade of the set point, that is, the incubation temperature programmed by a user, the control program starts a timer. This timer can be reset to count down from a number set as the time for the user-specified interval for the incubation in progress. The timer starts counting down from the preset value when the calculated sample temperature is within one degree centigrade. When the timer reaches a zero value, a signal is activated that causes the CPU to take actions to increment the next segment of the PCR protocol. Any way of timing the specified interval will suffice for the purpose of practicing the invention.

Typically, the tolerance band around any particular target temperature is plus or minus 0.5 ° C. Once the target temperature is reached, the calculator maintains the sample block at the target temperature using the control cooling channels and the film heater so that the samples remain near the target temperature for the specified interval.

For the thermal system described here to work well, the thermal conductivity from the sample block to each sample must be known and uniform within a very close tolerance. Otherwise, not all samples will stay within the tolerance band specified for the target temperature when the timer starts, and not all samples will experience the same incubation intervals at the target temperature.

Also, for this thermal system to work well, all sample tubes must be isolated from variables in the ambient environment. That is, it is undesirable for certain sample tubes to be cooled by air currents while other sample tubes in different physical positions do not experience the same cooling effects. To achieve good uniformity it is highly desirable that the temperatures of all samples be determined by the temperature of the sample block and nothing else.

Isolation of the tubes from the environment and the application of a minimal level of downward force F on the sample tubes is achieved by a heated cover over the sample tubes and the sample block.

Even though the liquid sample is in a sample tube tightly pressed against a temperature controlled metal block, with a tight seal, with a meniscus well below the surface of the temperature controlled metal block, the samples will still lose their heat. upwards by convection. Significantly, when the sample is very hot (the denaturation temperature is typically near the boiling point of the liquid sample), the liquid sample can lose a very significant amount of heat to the reflux of water vapor. In this process, water evaporates from the hot surface of the liquid sample and condenses on the inside of the lid walls and cold tops of the sample tube above the top surface of the sample block. If there is a relatively large sample volume, condensation continues and condensate forms upstream that runs down the walls of the sample tube into the reaction mixture. This "reflux" process takes about 2300 joules of heat per gram of refluxing water. This process can produce a several degree drop in the surface temperature of a 100 microliter reaction mixture thereby causing a large reduction in reaction efficiency.

If the reaction mixture is small, say 20 microliters, and the sample tube has a relatively large surface area above the top surface of the sample block, a significant fraction of the water in the reaction mixture may evaporate. This water can then condense on the inside of the top of the mixing tube and remain there by surface tension for the remainder of the high temperature part of the cycle. This can so concentrate the remainder of the reaction mixture that the reaction is impaired or fails completely.

In prior art PCR thermal cyclists, the reflux problem was solved by coating the reaction mixture with a layer of molten wax or oil. This immiscible layer of oil or wax floated in the aqueous reaction mixture and prevented rapid evaporation. However, work was required to add the oil which raised the cost of the processes. Additionally, the presence of oil interfered with subsequent processing and analysis steps and created a possibility of contamination in the sample. In fact, industrial grade mineral oils have been known to contaminate samples in the past due to the unknown presence of contaminating factors in the oil that were unknown to users.

The need for an oil coating is eliminated, and the problems of heat loss and concentration of the reaction mixture by evaporation and unpredictable thermal effects of reflux are avoided, in accordance with the teachings of the invention, by enclosing the volume. above the sample block into which they are projected

ES 2 318 232 T3 the upper parts of the sample tube and heating this volume from above by a heated cover sometimes also referred to hereinafter as the pressure plate.

Referring to Figure 19, there is shown a sectional view of the structure used to enclose the sample tubes and apply a downward force on them such that the minimum level of force F of Figure 15 is supplied. A heated pressure plate 14 is attached to a load screw 312 so that it moves up and down along the axis symbolized by arrow 314 with the rotation of the load screw. The charging screw is threaded through an opening in the sliding cover 316 and is rotated by a knob 318. The pressure plate 314 is maintained at a temperature above the boiling point of water by resistance heaters (not shown). computer controlled 20.

The sliding cover slides back and forth along the Y axis on the rails 320 and 322. The cover 316 includes the vertical sides 317 and 319 and also includes the vertical sides parallel to the XZ plane (not shown) that enclose the block of samples 12 and sample tubes. This structure basically prevents air currents from acting on the sample tubes, of which tubes 324 and 326 are typical.

Figure 20 is a perspective view of slide cover 316 and sample block 12 with the slide cover in the removed position to allow access to the sample block. The slide cover 316 resembles the lid of a rectangular box with the wall 328 having the portion 330 removed to allow the slide cover 316 to slide over the sample block. The sliding cover is moved along the Y axis in Figure 20 until the cover is centered over the sample block 12. The user then turns the knob 318 in one direction to lower the heated pressure plate 14 until a mark 332 on knob 318 aligns with a mark 334 on gusset 336. In some embodiments, gusset 336 may be permanently attached to the top of slide cover 316. In other embodiments. The gusset 336 can be rotated so that the mark 334 can be positioned in different positions when using different sizes of sample tubes. In other words, if taller sample tubes are used the heated pressure plate 14 does not need to be lowered as much to apply the minimum level of force F of Figure 15. In use, the user turns screw 318 to lower the pressure plate. 14 until the index marks line up. The user then knows that the minimum force level F has been applied to each sample tube.

Referring together to Figures 15 and 19, prior to lowering the heated pressure plate 14 of Figure 19, the plastic caps 338 of each sample tube protrude about 0.5 mm above the level of the top of the walls. of plastic tray 340 (Figure 19) that holds all sample tubes in a separate 8 x 12 matrix with centers at 9 millimeters. The sample well array can hold up to 96 100 μl capacity MicroAmp ™ pCr tubes or 48 larger 0.5 ml capacity GeneAmp ™ tubes. The details of this tray will be discussed in great detail below. Tray 340 has a flat surface with an 8 x 12 hole pattern for sample tubes. This flat surface is shown in Figures 15 and 19 as a horizontal line that intersects the sample tubes 324 and 326 in Figure 19. The tray 340 also has four vertical walls two of which are shown at 342 and 344 of Figure 19. The upper level of these vertical walls, shown at 346 of Figure 15, establishes a rectangular box defining a reference plane.

As best seen in Figure 15, the caps 338 of all sample tubes project above this reference plane 346 by a small amount that is designed to allow the caps 338 to be softened and deformed by the pressure plate. heated 14 and "squashed" down to the level of reference plane 346. In the preferred embodiment, heated pressure plate 14 is maintained at a temperature of 105 ° C by CPU 20 of Figure 1 and bus 22 coupled to resistor heaters (not shown) on pressure plate 14. In In the preferred embodiment, the knob 318 of FIG. 19 and the charging screw 312 are turned until the heated pressure plate 14 descends and contacts the tops of the caps 338. In the preferred embodiment, the caps 338 of the sample tubes are made of polypropylene. These caps soften shortly after coming into contact with the heated pressure plate 14. As the caps soften, they deform, but do not lose all their elasticity. After contacting the caps, the heated pressure plate is lowered further until it rests on reference plane 346. This further lowering deforms the caps 338 and produces a minimum level of force F of at least 50 grams to push each sample tube and keep each tube firmly seated in its sample well. The amount by which the caps 338 project above the reference plane 346, and the amount of residual deformation and elasticity when the heated pressure plate 14 rests on the reference plane 346, are designed such that a level has been reached. minimum force F of at least 50 grams and preferably 100 grams for all sample tubes then present after the heated pressure plate 14 has lowered to the level of the reference plane 346.

The heated pressure plate 14 and the four vertical walls and flat surface of the tray 340 form a heated compartment, sealed when the pressure plate 14 is in contact with the upper edge 346 of the tray. The plastic in the tray 340 has a relatively poor thermal conductivity property. It has been found experimentally that by contacting the heated pressure plate 14 with the caps 338 and isolating the portion of the sample tubes 288 that project above the upper level 280 of the sample block 12 with a wall of material having a Relatively poor thermal conductivity has beneficial results. With this structure, the entire top of the tube and cap are brought to a temperature that is high enough that little or no condensation forms on the inner surfaces of the tube and cap as the heated plate is maintained at a temperature above the boiling point of water. This is true even when the sample liquid

ES 2 318 232 T3

276 of Figure 15 is heated to a temperature near its boiling point. This eliminates the need for a layer of immiscible material such as oil or wax floating on top of the sample mixture 276 and thereby reducing the amount of work involved in the PCR reaction and eliminating a source of possible sample contamination.

It has been found experimentally that despite the very high temperature of the heated jacket and its close proximity to the sample block 12, the effect on the ability of the sample block 12 to cycle accurately and rapidly between high and low temperatures is small.

The heated pressure plate 14 prevents cooling of the samples by the aforementioned reflux process because it keeps the temperature of the lids above the dew point of water and hence the interiors of the lids are kept dry. This also prevents the formation of vapors when the caps are removed from the tubes.

In alternative embodiments, any means by which the minimum acceptable downward force F of Figure 15 can be applied to each individual sample tube regardless of the number of sample tubes present and which can prevent condensation and reflux and convection cooling will suffice. for the purposes of practicing the invention. The application of this downward force F and the use of heat to prevent reflux and unwanted concentration of the liquid sample need not both be implemented by the same system that is used in the preferred embodiment.

Sample tubes can vary by a few hundredths of a millimeter in their total height. Additionally, sample tube caps can also vary in height by a few hundredths of a millimeter. Also, each conical sample well in the sample block 12 may not be drilled to exactly the same depth, and each conical sample well in the sample block may be drilled at a slightly different diameter and angle. Thus, when a population of capped tubes is placed in the sample block to settle into corresponding sample wells, the tops of the caps will not necessarily all be at the same height. The worst case discrepancy for this height could be as much as 0.5 millimeters from the highest to the lowest of the tubes.

If a perfectly flat, unheated pressure plate 14 mounted so that it is free to find its own position were to be pressed down on such an array of caps, it would touch the three tallest tubes first. By applying additional pressure and the higher tubes being compressed in some way, the pressure plate would start to touch some of the lower tube caps. There is a distinct possibility that unless the tube and cap assembly was fitted, the taller tubes would be damaged before the lower tubes were in contact at all. Alternatively, the force necessary to compress all the tall tubes sufficiently to contact the shorter tubes should be too great to be applied to the device. In either case, one or more short tubes could not be pressed at all or could be pressed down with an insufficient amount of force to ensure that the thermal time constant for that tube was the same as the thermal time constants for all remaining tubes. This would cause the failure to achieve the same PCR cycle for all tubes in the sample block since the same tubes with different thermal time constants would not follow the same step as the rest of the tubes. Heating the pressure plate and softening the caps eliminates these risks by preventing them from being a factor in manufacturing tolerance errors that lead to different tube heights.

In an alternative embodiment, the entire heated pressure plate 14 is covered with a tailored rubber layer. A tailored rubber layer over the heated pressure plate would solve the height tolerance problem, but could also act as a thermal insulation layer that would retard the flow of heat from the heated pressure plate to the tube caps. Additionally, after long use at high temperatures, most rubber materials deteriorate or become hard. It is therefore desirable that the surface of the heated pressure plate be metal and a good conductor of heat.

In another alternative embodiment, 96 individual springs can be mounted on the pressure plate so that each spring individually presses down on a single sample tube. This is a complex and expensive solution, however, and requires the pressure plate to be aligned over the tube array with mechanical precision that would be difficult or cumbersome to achieve.

The individual adaptation required for each sample tube in the preferred embodiment is provided by the use of plastic caps that collapse predictably under the force of the pressure plate but which, even when collapsed, still exert a downward force F on sample tubes that is suitable for keeping each sample tube firmly seated in its well.

In the cap of the sample tube 338 shown in Figure 15, the surface 350 should be free of indentations, smudges and shaped that can provide a tight seal with the interior walls 352 of the sample, tube 288. In the preferred embodiment, the material of the cap is polypropylene. A suitable material could be Valtec HH444 or PD701 polypropylene manufactured by Himont as described above or PPW 1780 from American Hoescht. In the preferred embodiment, the wall thickness for the domed portion of the lid is 0.330 + 0.00-0.013 cm (0.130 + 0.000-0.005 inches). The thickness of the shoulder portion 356 is 0.6 mm (0.025 inches) and the width of the dome-shaped cap portion is 0.52 cm (0.203 inches) in the preferred embodiment.

ES 2 318 232 T3

Any cap material and configuration that causes the minimum level of force F in Figure 15 to be applied to all sample tubes and allows the cap and tops of the sample tubes to heat up to a sufficiently high temperature to prevent condensation and reflux will suffice for the purposes of practicing the invention. The domed cap 338 has a thin wall to aid in cap deformation. Because the heated pressure plate is kept at a high temperature, the wall thickness of the dome-shaped lid can be thick enough to be easily manufactured by injection molding as the necessary adaptation to account for differences at the height of the tubes is not necessary at room temperature.

The pressure plate is maintained at a temperature anywhere between 94 ° C to 110 ° C in accordance with the teachings of the invention although the range of 100 ° C to 110 ° C is preferred to prevent reflux since the point of boiling water is 100 ° C. In this temperature range, the caps have been experimentally found to soften just enough to easily collapse by up to 1 millimeter. Studies have shown that the elastic properties of the polypropylene used are such that even at these temperatures, the collapse is not entirely inelastic. That is, even though the heated pressure plate causes permanent deformation of the caps, the cap material still retains a significant enough fraction of its elasticity at room temperature so that the minimum level of force F is applied to each tube of sample. Additionally, the heated pressure plate levels all caps it contacts without excessive force regardless of how many tubes are present in the sample block due to softening of the cap.

Because the temperature of the cap is above the boiling point of water throughout the PCR cycle, the interior surfaces on each cap remain completely dry. Thus, at the end of the PCR process, if the samples are cooled to room temperature before being removed from the sample block, if the lid of each sample tube is opened, there is no possibility of creating a vapor spray from the contents of the tube. of samples that could result in cross contamination. This is because there is no liquid in the cap that seals the tube when the seal is broken.

This is extremely advantageous, because tiny vapor particles, containing amplified DNA product can contaminate a laboratory and reach sample tubes containing samples from other sources, for example, other patients, possibly thereby causing false positive or negative diagnostic results that they can be very disturbing. Users of PCR amplification processes are extremely concerned that vapors are not created that could contaminate other samples.

A system of disposable plastic elements is used to convert individual sample tubes into an 8 x 12 array that is compatible with laboratory equipment in microtiter plate format but maintains sufficient individual freedom of movement to compensate for differences in measurements. various rates of thermal expansion of system components. The relationship of the thermally matched cap to the rest of this system is best seen in Figure 21A which is a sectional view of the sample block and two sample tubes with caps in place with the sample tubes in place by the combination of one embodiment of a 96-well plastic microtiter tray and a retainer. Figure 21B is an alternative, preferred embodiment showing the structure and interrelationship of most of the various disposable plastic components of the system. The 96-well rectangular plastic microtiter plate tray 342 rests on the surface of the sample block 12. The upper edge 346 of the frame 342 at a height that is approximately 0.5 mm shorter than the height of the lids of which cap 364 is the example. All capped tubes will project their height beyond edge 346 of frame 342. Frame 342 is configured such that a shoulder 366 extends downward and engages the groove in guard band 78 along its length. total. The frame 342 does however have a notch (not shown) that corresponds to the notch in the slot 78 for the temperature sensor shown in Figure 2 seen in plan and in Figure 7 seen in section.

The aforementioned reference plane 346 is established by the top of frame 342. This reference plane interacts with the heated pressure plate as follows. Before turning the knob 318 of figure 20 down to align the index marks 332 and 334 and begin to perform an amplification, a calibration process has been performed to locate the position of the index marked on the escutcheon plate 336 of the figure. twenty. This calibration is initiated by placing the frame 342 of Figure 21 in position on the sample block. The frame 342 will however be empty or either of the sample tubes there will have none of their caps attached. The knob 318 is then turned downward until the heated pressure plate 14 is firmly in contact with the upper edge 346 of frame 342 around its entire perimeter. When the knob 318 has been turned down enough to allow the heated pressure plate to rest on the reference plane 346 and press the frame 342 firmly against the upper surface 280 of the sample block, the rotatable escutcheon 336 of the preferred embodiment will be rotated until index mark 334 on escutcheon lines up with index mark 332 on knob 318. Knob 318 is then turned counterclockwise to raise pressure plate 14 and cap 316 in Figure 19 is slid in the negative Y direction to reveal frame 342 and sample block 12. Sample tubes with caps in place and filled with a sample mixture can then be placed in position within frame 342. The heated cover 316 is placed back on the sample block, and the knob 318 is turned clockwise to lower the heated pressure plate 14 until the index mark 332 on the knob aligns with the mark. previously positioned index 334. This ensures that all tubes have been firmly seated with the minimum force F applied. The use of index marks provides the user with a simple and verifiable task to perform.

ES 2 318 232 T3

If there are only a few sample tubes located, only a small amount of twisting force will be needed to align the index marks 332 and 334. If there are many tubes, however, more twisting force will be needed on knob 318 to align the index marks. This is because each tube resists the downward movement of the heated pressure plate 14 as the caps deform. However, the user ensures that when the index marks 332 and 334 are aligned, the heated pressure plate will once again be positioned firmly against the top edge 346 of frame 342 and all tubes will have the minimum level of force F applied on them. This virtually guarantees that the thermal time constant will be substantially the same for all tubes.

In alternative embodiments, the index marks 332 and 334 can be dispensed with, and the knob 318 can simply be turned clockwise until it turns no more. This condition will occur when the heated pressure plate 314 has reached the upper edge of the reference plane 346 and the plastic frame 342 has stopped further downward movement of the heated pressure plate 14. Obviously in this alternative embodiment, and preferably in the embodiment of the index mark described above, the plastic of the frame 342 will have a melting temperature that will be high enough to prevent deformation of the plastic of the frame 342 when it is in contact with the plate. heated pressure 14. In the preferred embodiment, the plastic of the frame 342 is Celanese nylon 1503 with a wall thickness of 1.27mm.

An advantage of the systems described above is that sample tubes of different heights can be used simply by using frames 342 having different heights. Frame 342 should have a height that is approximately 0.5mm shorter than the plane of the tips of the capped tubes when both are seated on the sample block. In the preferred embodiment, two different tube heights are used. The range of movement of the pressure screw 312 that actuates the heated pressure plate 14 of Figure 19 should be sufficient for all sizes of sample tubes to be used. Of course, during any particular PCR run, all tubes must be the same height.

The above-described system provides uniform temperatures across the sample block, uniform thermal conductivity from the sample block, and isolation of the sample tubes from the vagaries of the surrounding environment. Any number of sample tubes up to 96 can be arrayed in microtiter plate format. The system allows precise temperature control for a large number of samples and visual indication of sample temperature for all samples without actually measuring the temperature of any sample.

As a container for PCR reactions, it has been common in prior art to use polypropylene tubes that were originally designed for microcentrifuges. This prior art tube had a cylindrical cut section closed at the top by a tight-fitting cap which performed gas sealing. This prior art tube had a lower section that compressed the trunk of a cone with an included angle of approximately 17 degrees.

When such a conical sample tube is pressed down into the sample well of a sample block with a conical cavity with the same included angle, and when the sample mixture in the tube falls entirely within the conical volume and below On the upper surface of the sample block, the thermal conductivity between the block and the liquid can be made suitably predictable for good uniformity of the sample temperature across the matrix. To achieve adequate control of thermal conductivity between the sample block and the sample mixture, the included angles of the conical tube and sample well must be very close, and the conical surfaces of the tube and well must be smooth and maintained. together in a relationship on a level. Additionally, the minimum level of force F must be applied to each sample tube to press each tube tightly into its sample well so that it does not lift or slack in the well during thermal cycling for any reason, such as formation of vapor in the liquid trapped in space 291 of figure 15. Finally, each tube must be filled with the same amount of liquid sample. If the conditions listed above are met, the thermal conductivity between the sample block and the liquid sample in each tube will be determined predominantly by the conductivity of the plastic wall of cone 368 of Figure 15 and a boundary layer (not shown) of the liquid sample on the inner surface 370 of the conical wall of the sample tube.

The thermal conductivity of the plastic tube walls is determined by its thickness, which can be rigorously controlled by the injection molding method in the manufacture of the tubes. The liquid sample in all sample tubes has virtually identical thermal properties.

A single 96-well microtiter plate molding has been found experimentally and by calculation to be only marginally viable for PCR because differences in thermal expansion coefficients between aluminum and plastic lead to dimensional changes that can destroy the uniformity of thermal conductivity with the liquid sample through the matrix. That is, since each well in such a single piece plate connects to the other wells across the surface of the plate, the distances between the wells are determined at the time of initial plate manufacture but change with temperature. changing since the plastic of the plate has a significant coefficient of thermal expansion. Also the distances between the sample wells in the metallic sample block 12 depend on the temperature of the sample block since aluminum also has a significant coefficient of thermal expansion that is different from plastic. To have good thermal conductivity, each sample well in a microtiter plate in a 96-well piece should fit almost perfectly to the corresponding well in the sample block at all temperatures. Since the temperature of the sample block changes over a wide temperature range, the distances between the

ES 2 318 232 T3 sample wells in the sample block vary cyclically during the PCR cycle. Because the coefficients of thermal expansion for plastic and aluminum are substantially different, the spacing distances of the wells in the sample block would vary differently with changing temperatures than would the distances between the sample wells of the one piece 96-well plastic microtiter plate.

Thus, as an important criterion for a perfect fit between the sample tube and the corresponding sample well in the PCR temperature range, it is necessary that each sample tube in the 96-well array is individually free to move laterally and that Each tube is individually free to be pressed down vertically in any amount necessary to make flush contact with the walls of the sample well.

The sample tubes used in the invention are different from prior art microcentrifuge tubes in that the thickness of the walls of the conical stem position of the sample tube is thinner to allow faster heat transfer to and from the sample tube. sample liquid. The top of these tubes has a thicker wall thickness than the tapered part. In Figure 15, the wall thickness in the cylindrical portion of Figure 15 is generally 0.76 mm (0.030 inches) while the wall thickness of the tapered wall 368 is 0.23 mm (0.009 inches). . Because thin parts cool faster than thick parts in the injection molding process, it is important to completely remove the mold before the thin parts cool down.

The material in the sample tubes must be chemically compatible with the PCR reaction. Glass is not a PCR compatible material, because DNA sticks to glass and will not peel off which would interfere with pCr amplification. An autoclavable polypropylene is preferably used. Here three suitable types of polypropylene were previously identified. Some plastics are not compatible with the PCR process due to the release of gaseous materials from the plastic or because DNA adheres to the plastic walls. Polypropylene is the best of the known plastics at the moment.

Conventional injection molding techniques and injection mold mold making techniques are sufficient for the purpose of practicing the invention.

Using cone-shaped sample tubes basically translates all manufacturing tolerance errors into height errors, that is, a tube-to-tube variation in height from the tip of the cap to the top of the block. samples when the sample tube sits in the sample well. For example, an angular error for the angle of the sample tube walls becomes a height error when the tube is placed in the sample block due to the mismatch between the angle of the tube wall and the angle of the wall. sample well. In the same way, a diameter error in the dimensions of the cone would also result in a height error since the tapered part of the tube would either penetrate deeper or not as deeply as an appropriately sized tube.

To have good uniformity in thermal conductivity across the array, there must be a good fit between the sample tubes and the sample wells in all 96 wells for the full range of temperatures from 0 to 100 ° C regardless of the temperatures. different rates of thermal expansion. Also, each of the 96 tubes must have walls with dimensions and wall thickness that are uniform to a high degree. Each sample tube in which the sample mixture is to be kept must be provided with a gas adjusting cap that provides a gas seal to prevent the loss of water vapor from the reaction mixture when this mixture is in or out. near the boiling point so that the volume of the sample mixture does not decrease. All of these factors combine to make a one-piece microtiter plate with 96 individual sample wells extremely difficult to fabricate so that uniform thermal conductivity is achieved for all 96 wells.

Any structure that provides the necessary individual lateral and vertical degrees of freedom for each sample tube will suffice for the purpose of practicing the invention.

In accordance with the teachings of the preferred embodiment of the invention, all of the above-mentioned requirements have been met using a four-piece disposable plastic system. This system gives each sample tube sufficient freedom of movement in all directions necessary to compensate for different rates of thermal expansion while still maintaining up to 96 sample tubes in a 96-well microtiter plate format for user convenience and compatibility. with other laboratory equipment that is sized to work with the industry standard 96-well microtiter plate. The disposable plastic multi-part system is very forgiving of manufacturing tolerance errors and different rates of thermal expansion over the wide temperature range encountered during thermal cycling of PCR.

Figures 21A and 21B show alternate embodiments of most components of the four-piece plastic system viewed in section as assembled to hold a variety of sample tubes in their sample wells with sufficient freedom of movement to account for the different rates of thermal expansion. Figure 45 shows all parts of the emulation system of a disposable plastic microtiter plate seen in exploded view. This figure illustrates how the parts fit together to form a microtiter plate with all sample tubes flexibly retained in a 96-well array in 8 x 12 microtiter plate format. Figure 22 shows a plan view of a microtiter plate frame 342 in accordance with the teachings of the invention which is shown partially in section in Figures 21A and 21B. Figure 23 shows a bottom plan view of frame 342. Figure 24 is an end view of frame 342 taken from the

ES 2 318 232 T3 view of line 24-24 'of Figure 22. Figure 25 is an end view of frame 342 taken from the point of view of line 25-25' of Figure 22. Figure 26 is a sectional view of frame 342 taken along line 26-26 'of Figure 22. Figure 27 is a sectional view of frame 342 taken along line 2727' of Figure 22. Figure 28 is an end view of frame 342 taken from along line 28-28 'of Figure 22 with a partial section to show in more detail the location where a retainer to be described later attaches to the frame 342.

Referring together to Figures 21A, 21B and 22-28, frame 342 is comprised of a horizontal plastic plate 372 in which 96 holes spaced 9mm between centers are reported in the standard microtiter plate format. There are eight rows labeled A through H and 12 columns labeled 1 through 12. Hole 374 in row D, column 7 is typical for these holes. A conical sample tube such as sample tube 376 shown in Figure 15 is placed in each hole of frame 342. Each sample tube is smaller in diameter than the hole in which it is located by about 0.7 mm. , so it fits loosely in the hole. This is best seen in Figures 21A and 21B by looking at the distance between the inner edge 378 of a typical hole and the side wall 380 of the sample tube located there. Reference numeral 382 in Figures 21A and 21B shows the opposite edge of the hole that is also spaced from the outer wall of the cylindrical portion of sample tube 376.

Each sample tube has a rim shown at 384 on fibers 15, 21, and 29. This rim is molded around the entire circumference of the cylindrical portion 288 of each sample tube. The diameter of this flange 384 is large enough that the tube does not pass through the holes in the frame 342. But not so great that the flanges touch those of the adjacent tubes in neighboring holes.

Once all the tubes are positioned in their holes in frame 342, a plastic retainer 386 (best seen in Figures 21A and 21B and Figure 45) is clipped over the openings in frame 342. The The purpose of this retainer is to hold all tubes in place so that they cannot fall or knock out of frame 342, while not interfering with their clearance to fit into frame 342. Retainer 366 is sized and adjusted to frame 342 such that each sample tube is free to move up and down to some extent before rim 384 of the tube meets either retainer 386 or ok with frame 342. Thus, the frame and retainer, when coupled, provide a microtiter plate format for up to 96 sample tubes but provide sufficient horizontal and vertical freedom such that each tube is free to find its best fit in all temperatures under the influence of the minimum force level F in figure 15.

A clearer view of the sample tube and its flange can be had with reference to Figures 29 and 30. Figures 29 and 30 are sectional elevational views and an upper partial section of the flange portion, respectively, of a typical sample tube. A dome-shaped cap such as that which will be described in more detail below is inserted into the sample tube shown in FIG. 29 and forms an airtight seal with the inner wall 390 of the top of the sample tube. A shoulder 392 formed on the inner wall of the sample tube acts as a stop for the dome-shaped cap to prevent further penetration. Typically, dome-shaped lids come in strips connected by a net.

Figure 31 shows three caps in elevation view connected by a net 394 and terminated in a tab 396. The tab assists the user in removing the entire row of caps with a simple movement. Typically, the net 394 rests on the top surface 398 of the sample tube and prevents further penetration of the cap into the sample tube. Each cap includes a shoulder 400 that forms a hermetic seal between the cap and the inner wall of the sample tube. Figure 32 shows a top view of the three caps of a typical strip of 12 attached caps.

For a more detailed understanding of the retainer, we refer to Figures 33 to 37. Figure 33 is a top view of the plastic retainer. In Figure 34 is an elevation view of the retainer taken along line 34-34 'in Figure 33. In Figure 35 is an elevation view of the retainer taken along line 35 -35 'in Figure 33. Figure 36 is a sectional view taken along line 36-36' in Figure 33. Figure 37 is a sectional view through the retainer taken along line 37-37 'in Figure 33.

Referring together to Figures 33-37, the retainer 386 is comprised of a single horizontal plastic plane 402 surrounded by a vertical wall 404. The plane 402 has 96 holes in an 8 x 12 matrix formed in it and divided into 24 groups of four holes per group. These groups are determined by the ridges formed in plane 402 such as ridges 406 and 408. Each hole, of which hole 410 is typical, has a diameter D that is greater than diameter D₁ in figure 29 and smaller than diameter D₂. This allows the retainer to slide over the sample tubes after they have been positioned in frame 342 but prevents the sample tubes from falling out of the frame as flange 384 is too large to pass through hole 410.

The retainer clips into frame 342 via plastic tabs 414 shown in Figures 34 and 36. These plastic tabs are inserted through slots 416 and 418 in the frame as shown in Figure 23. There are two plastic tabs 414, one on each long edge of the retainer. These two plastic tabs are shown as 414A and 414B in Figure 33.

ES 2 318 232 T3

The frame 342 of Figures 22-28, with up to 96 sample tubes positioned in it and with the retainer 386 snapped in place, forms a single assembly as shown in Figures 21A and 21B which can be positioned over sample block 12 for PCR processing.

After processing, all tubes can be removed simultaneously by lifting frame 342 from the sample block. For convenience and storage, the frame 342 with the sample tubes and retainer in place can be inserted into another plastic component called the base. The base has the outer dimensions and footprint of a standard 96-well microtiter plate as shown in Figures 38 to 44. Figure 38 is a top plan view of the base 420, while Figure 39 is a bottom plan view of the base. Figure 40 is an elevational view of the base taken from line 40-40 'in Figure 38. Figure 41 is an end elevational view taken from line 41-41' in Figure 38. Figure 42 is a sectional view through the base along line 42-42 'in Figure 38. Figure 43 is a sectional view through the base along line 43-43 'in Figure 38. Figure 44 is a sectional view along line 44-44' in Figure 38 .

Base 420 includes a flat plastic surface 422 in which an 8x12 hole pattern with sloping edges is formed. These holes are dimensioned and spaced such that when frame 342 sits on the base, the bottom of the sample tubes snap into the conical holes in the base so that the sample tubes are positioned relative to each other. frame 342 in the same manner as when the sample tubes are positioned when frame 342 is mounted on the sample block. The hole 424 is typical of the 96 objects formed in the base and is shown in Figures 38, 44 and 43. The individual sample tubes, despite being loosely retained between the tray and the retention element, remain firmly seated and immobile when the frame is inserted into the base. The manner in which the typical sample tube 424 fits into the base is shown in figure 44.

In other words, when the frame, sample tubes, and retainer are seated in the base 420, the assembly becomes the exact functional equivalent of an industry standard 96-well microtiter plate, and can be placed in virtually any system. Automatic sampling or pipetting for industrial standard 96-well microtiter plates for other processes.

After the sample tubes have been filled with the necessary reagents and the DNA sample to be amplified, the sample tubes can be capped. In an alternative embodiment of the strip of caps shown in Figures 31 and 32, an already complete mat of 96 caps with a matched net may be used by connecting them in an 8 x 12 matrix. This network, shown at 394 of figure 31, must be sufficiently adapted so that the caps do not prevent the sample tubes from making the small movements that these tubes must perform to fit perfectly in the conical wells of the sample block at all temperatures.

The set of tubes, cap frames, retainer and base is taken after filling the tubes to the thermal cycle maker. There, the frame, the capped tubes and the retainer plate are separated from the base as a unit. This unit is then positioned in the sample block 12 to perform the assembly shown in Figure 21A or 21B with the tubes loosely contained in the conical wells in the sample block. As shown in Figure 21, frame 342 sits on top surface 280 of the guard strip. In the preferred embodiment, the shoulder 366 extends down into the groove 78 of the guard band, but this is not essential.

The heated cap is then slid over the samples, and the heated pressure plate is screwed down as described above until it contacts the top edge 346 of frame 342.

A few seconds after the heated pressure plate 14 of Figure 19 touches the caps, the caps begin to soften and yield under the downward pressure of the charging screw 312 of Figure 19. The user then continues to turn the knob 318 until the indices 332 and 334 in Figure 20 align which indicates that each sample tube has been tightly pressed into the sample block with at least the minimum level of force F. and that all of the air spaces between the heated pressure plate 14, the sample block, and the upper edge 346 of the frame 342 have been tightly closed. The sample tubes are now in a completely closed and controlled environment, and you can accurately cycle temperatures.

At the end of the PCR protocol, the heated pressure plate 14 is moved up and out of the sample tubes, and the heated cover 316 is slid to expose the frame 342 and the sample tubes. The frame, sample tubes, and retention element are then removed and repositioned in the empty base, and the caps can be removed. As each cap or strip of caps is pulled out, the retainer holds the tube by preventing it from exiting the tray. Ribs formed in the base (not shown in Figures 38-44) contact the retainer tabs 414A and 414B shown in Figure 33 to hold the retainer locked in place so that the force exerted on the Tubes removing caps does not dislodge retainer 386.

Obviously, the 342 frame can be used with fewer than 96 tubes if desired. Also, the retainer 386 can be removed if desired by disengaging it.

A user who wants to work with only a few tubes at a time and handle these tubes individually should place an empty frame 342 without a retainer on the sample block. The user can then use the base as a "test tube rack" and leave a small number of tubes in it. These tubes can then be filled

ES 2 318 232 T3 manually and capped with individual caps. The user can then transfer the tubes individually into the wells of the sample block, close the heated cover, and screw on the heated pressure plate 14 until the markings line up. The PCR cycle can then begin. When the cycle is complete, the cover 316 is removed and the sample tubes are individually placed on a disposable base. The retention element is not necessary in this type of use.

Referring to Figures 47A and 47B (hereinafter Figure 47), there is shown a block diagram for the electronics of a preferred embodiment of a control system in a type of control system represented by the CPU block 20 of Figure 1. The purpose of the control electronics of figure 47 is, among other things, to receive and store the data entered by the user defining the desired PCR protocol, read the various temperature detectors, calculate the temperature of the sample, compare the temperature of the sample. sample calculated with desired temperature for user-defined PCR protocol, monitor the power line voltage and control the film heater zones and rapid cooling valves to perform the desired temperature profile of the user defined PCR protocol.

A microprocessor (hereinafter CPU) 450 executes the control program described below cited in Appendix C in the form of source code. In the preferred embodiment, CPU 450 is an OKI CMOS 8085. The CPU controls an address bus 452 through which several of the other elements of the circuit of Figure 47 are addressed. The CPU also controls a data bus 454 to through which the data is transmitted to several of the other elements of the circuit of Figure 47.

The control program in Appendix C and some of the system constants are stored in the EPROM 456. User-entered data and other system constants and characteristics measured during the installation process (execution of the installation program described below) they are stored in battery powered RAM 458. A clock / calendar 460 provides the CPU 450 with date and time information in order to record a history of events during PCR runs and the duration of power failures as described below in the control program description. .

An address decoder 462 receives and decodes the addresses from the address bus 452 and activates the appropriate circuit selection lines on the circuit selection bus 464.

The user enters the PCR protocol data through the keyboard 466 in response to the information displayed by the CPU on the screen 468. The two forms of communication between the user and the CPU 450 are described in more detail below in the interface section. with the user of the control program description. An interface circuit 470 with the keyboard converts the user's keystrokes into data that is read by the CPU via data bus 454.

Two programmable interval timers 472 and 474 each contain counters that are loaded with values ​​calculated by CPU 450 to control the intervals during which power is applied to the various zones of the film heater.

An interrupt controller 476 sends interrupt requests to CPU 450 every 200 milliseconds, causing CPU 450 to execute the PID task described below in the description of the control program. This task reads the temperature detectors and calculates the heating or cooling power required to bring the sample temperature from its current level to the level desired by the user for that point in time of the PCR protocol being run.

A UART 478 serves an RS232 interface circuit 480 so that data stored in RAM 480 can be sent to the printer. The control program maintains a record of each PCR run that is performed in relation to the actual temperatures that existed at various times during the run in order for the user to validate that the PCR protocol actually run corresponds to the PCR protocol desired by the user. . In addition, data entered by the user defining the desired times and temperatures during a particular PCR protocol is also stored. All this data as well as other data can be read by the CPU 450 and sent to a printer coupled to the RS232 port via the UART 478. The RS232 interface also allows an external computer to take control of the address and data buses. for testing purposes.

A peripheral interface circuit (PIC) 482 serves as a programmable set of four input / output registers. At startup, CPU 450 selects PIC 482 through address decoder 462 and circuit selection bus 464. The CPU then writes a data word to the PIC via data bus 454 to program the PIC 482 in relation to which registers are to be output ports and which are to be input ports. Subsequently, CPU 450 uses the output registers to store data words written to them by the CPU via data bus 454 to control the internal logic state of a programmable logic matrix (PAL) circuit 484.

The PAL 484 is a state machine that has a variety of input signals and a variety of output signals. PALs generally contain a logical array that has a number of different states. Each state is defined by the matrix or vector of logic states at the inputs and each state results in a different matrix or vector of logic states at the outputs. CPU 450, PIC 482, PAL 484 and various other circuits to be defined

ES 2 318 232 T3 then cooperate to generate different states of the various output signals of the PAL 484. These different states and associated output signals are what control the operation of the electronics shown in Figure 47 as will be described below.

A 12-bit analog to digital (A / D) converter 486 converts the analog voltages on lines 488 and 490 into digital signals on the data bus 454. These are read by the CPU by generating an address for the A / D converter. D so that a signal is activated on the 464 bus circuit selector coupled to the select input of the A / D converter circuit and activates the converter. The analog signals on lines 488 and 490 are the output lines of two multiplexers 492 and 494. Multiplexer 492 has four input ports, each having two signal lines. Each of these ports is coupled to one of the four temperature detectors in the system. The first port attaches to the sample block temperature detector. The second and third ports are coupled to the refrigerant and ambient temperature sensors, respectively, and the fourth port is coupled to the heated shell temperature sensor. A typical circuit for each of these temperature detectors is shown in Figure 48. A 20,000 ohm resistor 496 receives at node 427 regulated +15 V from regulated power supply 498 of Figure 47 through a power line. connection that is not displayed. This 15 Vdc signal reverse biases a zener diode 500. The reverse bias current and the voltage drop across the zener diode are a function of temperature. The voltage drop across the diode is input to multiplexer 292 via lines 502 and 504. Each temperature detector has a similar connection to multiplexer 292.

Multiplexer 494 also has four input ports but only three are connected. The first input port is coupled to a calibration voltage generator 506. This voltage generator draws two precisely controlled voltage levels for the multiplexer inputs and is thermally very stable. That is, the reference voltage output from voltage source 506 has a very small drift, if any, with temperature. This voltage is read from time to time by the CPU 450 and compared to a stored constant representing the level this reference voltage had at a known temperature as measured during the execution of the installation process described below. If the reference voltage has drifted from the measured and stored level during the installation process, the CPU 450 knows that all other electronic circuits used to detect the various temperatures and line voltages have also drifted and adjusts its outputs accordingly to maintain very precise control over the temperature measurement process.

The other input to multiplexer 494 is coupled through line 510 to an effective value to direct voltage converter circuit 512. This circuit has an input 514 coupled to a voltage drop transformer 516 and receives an alternating voltage at input 514 which is proportional to the line voltage then existing at the AC power input 518. The RMS to DC converter 512 rectifies the AC voltage and averages to have a DC voltage on line 510 that is also proportional to the AC input voltage on line 518.

Four optically coupled triac drivers 530, 532, 534, and 536 receive the input control signals via control bus 538 from logic pAl 484. Each of the triac drivers 530, 532, and 534 controls power at a of the three zones of the film heater. These heater zones are represented by blocks 254, 260/262, and 256/258 (the same reference numerals used in Figure 13). Triac actuator 536 controls power to the heated cover, represented by block 544 through thermal cutoff switch 546. The heater zones of the film heater are protected by a block of thermal cutoff switches 548. The purpose of the thermal cut-off switches is to prevent the film heater / sample block from melting onto the heated cover in the event that a failure leads to the triac actuators being left on for an unsafe interval. If such a situation occurs, the thermal cut-out switches will detect an over-hot condition, and will cut off the triacs via the 552 or 554 signals.

The zone heater main heater zone is rated at 360 watts while the manifold and edge heater zones are rated at 180 watts and 170 watts respectively. The actuators are Motorola MAC 15A10 15A triacs. Each zone of the heater is divided into two electrically isolated sections each dissipating 1/2 of the power. The two halves are connected in parallel for line voltages in 518 less than 150 V ac For line voltages higher than this, the two halves are connected in series. These alternate connections are made through a "personalization" connector 550.

The AC voltage supply for the film heater zones is line 559, and the AC supply for the heated cover is via line 560.

A zero crossing detector 566 provides basic system timing by emitting a pulse on line 568 at each zero crossing of the alternating voltage of line 518. The zero crossing detector is a National IX 311N referred to the ground voltage and has 25 mV hysteresis. The zero crossing detector takes its input from transformer 516 which obtains a 0 to 5.52 V ac signal for a 0 to 240 V ac input ac signal.

A power transformer 570 supplies the alternating power to the pump 41 which pumps refrigerant through the fast cooling and control channels. The cooling unit 40 also receives its AC power from the transformer 570 through another part of the customization connector 550. The transformer 350 also

ES 2 318 232 T3 supplies power to the three regulated power supplies 572, 498 and 574 and to an unregulated power supply 576.

For accuracy purposes in measuring temperatures, the calibration voltage generator 506 uses a series of very precise, thin film, ultra-low temperature drift resistors of 20 kQ (not shown in Figure 47).

These same ultra-low drift resistors are used to adjust the gain of an analog amplifier 578 that amplifies the output voltage of the selected temperature detector prior to converting it to a digital value. These resistors drift only 5 ppm / ° C.

All temperature detectors are calibrated by placing them (separate from the structures whose temperatures they measure) first in a stable bath, with stirring oil, temperature controlled at 40 ° C and measuring the actual output voltages at the inputs to multiplexer 492. Temperature detectors are then placed in a bath at a temperature of 95 ° C and their output voltages are measured again at the same points. The output voltage of the calibration voltage generator 506 is also measured at the input of the multiplexer 494. For each temperature, the difference of the digital output of the A / D converter 486 is measured between each of the outputs of the temperature detector and the digital output resulting from the voltage generated by the calibration voltage generator 506. The calibration constants for each temperature detector can then be calculated to calibrate each for changes in temperature.

The sample block temperature detector is then subjected to an additional calibration procedure. This procedure involves bringing the sample block to two different temperatures. At each temperature level, the actual temperature of the block in its different sample wells is measured using the thermocouple probe RTD 16 with an accuracy of 0.02 ° C. A profile of the mean block temperatures is then generated and the output of the A / D converter 464 is measured with the block temperature detector in place of the sample block. This is done at both temperature levels. From the actual block temperature as measured by the RTD probes and the A / D output of the block temperature detector, an additional calibration factor can be calculated. The temperature calibration factors thus generated are stored in battery-powered RAM 458. Once these calibration factors are determined for the system, it is important that the system does not appreciably derive its electrical characteristics from those existing at the time of installation. calibration. It is therefore important that low drift circuits are selected and that ultra low drift resistors are used.

How the CPU 450 controls the temperature of the sample block can be better understood by referring to the next section that describes the control program. However, to illustrate how the electronic circuits of Figure 47 cooperate with the control program to carry out the PCR protocol consider the following.

Zero crossing detector 566 has two outputs on output bus 568. One of these outputs emits a negative pulse for each positive transition of the AC signal as it passes the zero voltage reference. The other emits a negative pulse at each negative transition of the AC signal as it passes through the zero voltage reference. These two pulses, typically shown at 580, define one full cycle or two half cycles. It is the pulse train on bus 561 that defines the 200 millisecond sample periods. For an alternating current of 60 cycles / second like that found in the US, 200 milliseconds contains 24 half cycles.

A typical sample period is displayed in Figure 49. Each mark in Figure 49 represents one half cycle. During each 200 ms sample period, the CPU 450 is calculating the amount of heating or cooling power required to maintain the sample block temperature at the incubation or user-defined setpoint temperature or to move the block temperature. to the new temperature depending on where you are on the PCR protocol time line of the particular sample period. The amount of power required in each zone of the film heater is converted into a number of half cycles in which the zone heater must remain off for the next 200 ms sample period. Just prior to the end of the current sample period in which these calculations are performed, CPU 450 addresses each of the four timers in programmable interval timer (PIT) 472. At each timer, the CPU writes the data that constitutes a "current" count representing the number of half cycles that the zone heater associated with that timer is to remain off in the next sample period. In FIG. 49, this data is written to the timers during the interval 590 just preceding the start time 592 of the next sample period. Suppose that a rapid ramp to denaturing temperature of 94 ° C is required by user set data for an interval that includes the sample interval between times 592 and 594. Consequently, the film heaters will be on the most of the period. Suppose the central zone heater is to be on for all but three of the half cycles during the sample period. In this case, CPU 450 writes a three to the PIT 472 counter associated with the central zone heater during interval 590. This write operation causes the timer to send a "disconnect" signal to the particular control line. from bus 593 that controls the central zone heater. This "disconnect" signal causes the PAL 484 to send a "disconnect" signal on a particular one of the signal lines of the bus 538 associated with the central zone. The triac driver 530 then turns off at the next zero crossing, that is, at time 592. The PIT receives a pulse train of the positive transition pulses on line 594 from PAL 484. These pulses are translations of PAL 484 of the zero crossing pulses on the two-line bus

ES 2 318 232 T3

568 to positive pulses on all zero crossing pulses on a line, that is, line 594. The timer in PIT 472 associated with the center zone of the film heater begins its countdown from its current value of 3 using the flags half cycle pulse on line 594 as your clock. At the end of the third half cycle, this timer reaches 0 and causes its output signal on bus line 592 to change state. This transition from low to high state is shown at 596 of Figure 49. This transition is communicated to PAL 484 causing it to change the state of the appropriate output signal on bus 538 to connect triac driver 530 in the third pass through zero. Note that by connecting the triacs in the zero crossing as is done in the preferred embodiment, it is avoided to cut off the high current flowing through an inductor (the conductor of the film heater). This minimizes the generation of radio frequency interference or other noise. Note that the technique of switching a portion of each half cycle on the film heater in accordance with the calculated amount of power required will also work as an alternative embodiment, but is not preferred due to the noise generated by this technique.

The other PIT timers 472 and 474 work in a similar manner to control the power applied to the other heater zones and the heated deck according to the power calculated by the CPU.

Fast cooling is controlled by CPU 450 directly through peripheral interface 482. When calculations of heating / cooling power performed during each sample period indicate that fast cooling power is required, CPU 450 targets the peripheral interface controller (PIC) 482. A data word is then written to the appropriate register to set the output line 600 high. This output line triggers a pair of monostable multivibrators 600 and 604 and causes each to emit a single pulse, on lines 606 and 608, respectively. Each of these pulses has peak currents just below 1 A and a pulse duration of approximately 100 ms. The purpose of these pulses is to forcefully activate the solenoid valve coil that controls the flow through the fast cooling channels to quickly open the fast cooling flow. The pulse on line 606 causes an actuator 610 to ground line 612 attached to one side of the solenoid coil 614 of one of the solenoid actuated valves. The other terminal of coil 614 connects to a "common" of power supply 616 to +24 Vdc of power supply 576. One of the shots 602 controls the opening of the refrigeration solenoid valve to flow in one direction, and the other shot 604 controls the solenoid-operated valve to flow in the opposite direction.

Simultaneously, the activation of the RCOOL signal on line 600 causes the activation of an actuator 618. This actuator grounds line 612 through a limiting resistor 620. The value of this current limiting resistor is such that the current flowing across line 622 is at least equal to the holding current needed to keep solenoid valve 614 open. Solenoid coils have transient characteristics that require large currents to open a solenoid-actuated valve but considerably less current to keep the valve open. When the 100 ms pulse on line 606 decays, actuator 612 directly ceases grounding line 612 leaving only ground connection through resistor 620 and actuator 618 to hold current.

The solenoid valve 614 controls the flow of the fast cooling refrigerant through the sample block only in 1/2 of the fast cooling tubes, that is, the tubes that carry the refrigerant in one direction through the sample block. Another solenoid actuated valve 624 controls the flow of refrigerant through the sample block in the opposite direction. This valve 624 is actuated in exactly the same way as solenoid actuated valve 614 by actuators 626 and 628, one shot on 604 and 608 line.

The need for rapid cooling is evaluated once each sample period. When the PID task of the control program determines by measurements of the block temperature, comparing it to the desired block temperature that fast cooling is no longer needed, the RCOOL signal on line 600 is deactivated. This is done by CPU 450 by addressing PIC 482 and writing data to it that reverses the state of the appropriate bit in the PIC 482 register that is connected to line 600.

The PIT 474 also has two other timers in it that time an interrupt to 20 Hz and a warm-up LED that provides a visible indication when the sample block is hot and unsafe to touch.

The system also includes a buzzer monostable 630 and a buzzer 632 to warn the user when an incorrect keystroke will be performed.

The programmable interrupt controller 476 is used to detect seven interrupts: Level 1-test; Level 2-20 Hz; Level 3-stream ready; Level 4-reception ready; Level 5-keyboard interrupt; Level 6 main heater on; and Level 7-crossing zero on the discharge line.

The peripheral interface controller 482 has four outputs (not shown) to control multiplexers 492 and 494. These signals MUX1 eN and MUX2 EN enable one or the other of the two multiplexers 492 and 494 while signals MUX 0 and MUX 1 control what channel is selected for input from amplifier 578. These signals are managed such that only one channel can be selected from the two multiplexers at any one time.

An RLTRIG * signal resets a 632 monostable timer for the heaters that disables the heaters through the activation of the TIMEOUT EN * signal to PIL 484 if the CPU fails. That is, the 632 monostable has

ES 2 318 232 T3 a predetermined interval that is what he waits after each trip before he activates the TIMEOUT EN * signal that disables all zone heaters. CPU 450 periodically executes a routine that addresses PIC 482 and writes data to the appropriate register to activate a signal on line 634 and trigger monostable 632. If CPU 450 fails for any reason and does not execute this routine, monostable timer 632 disables all zone heaters.

The PIC 482 also has COVHTR EN * and BLKHTREN * outputs (not shown) to enable the heated cover and sample block heater. Both signals are active low and are controlled by the CPU 450. They are sent to the PAL 484 through the 636 bus.

The PIC 482 also supplies the BEEP and BEEPCLR * signals on the 640 bus to control the monostable of the buzzer 630.

The PIC 482 also supplies the MEM1 signal (not shown) which is used to switch pages between the high address section of the EPROM 456 and the low address section of the battery powered RAM 458. Two other PAGE SEL signals are provided. 0 and PAGE SEL 1 (not shown) to select from the four 16K pages in the EPROM 456.

The four temperature detectors are of the National LM 135 zener diode type with a zener voltage / temperature dependence of 10 mV / K. The zener diodes are driven by regulated power supply 498 through a 20K resistor 496. The current through the zeners ranges from approximately 560 µA to 615 pA in the operating range of 0 ° C to 100 ° C. Zener self heating ranges from 1.68 mW to 2.10 mW in the same range.

Multiplexers 492 and 494 are DG409 analog switches. The voltages on lines 488 and 490 are amplified by an AD625KN instrumentation amplifier with a transfer function of V_out = 3 * V_in 7.5. The 486 A / D converter is an AD7672 with a 0-5 volt input range. With the zener temperature detector output 2.73 to 3.73 volts in the 0 ° C to 100 ° C range, the output of the 578 amplifier will be 0.69 volts to 3.69 volts, which is comfortably within the range A / D input.

The key to high precision in system performance are good precision and low drift with changes in ambient temperature. Both objectives are achieved by using a precision voltage reference source, that is, the 506 calibration voltage generator, and continuously monitoring its output through the same electronic chain as that used to monitor the outputs of the temperature detectors already. AC line voltage on line 510.

The calibration voltage generator 506 provides two precision voltages on lines 650 and 652. One voltage is 3.75 V and the other 3.125 V. These voltages are obtained by dividing the regulated supply voltage using a chain of resistors of Integrated, ultra-low-drift, thin film film with 0.05% resistor fit and a temperature drift coefficient of 5ppm / degree Celsius between resistors. The calibration voltage generator also outputs -5 V for the A / D converter voltage reference and -7.5 V for the instrumentation amplifier compensation. These two voltages are connected to A / D 486 and amplifier 578 on lines not shown. These two negative voltages are generated using the same network of thin film resistors and an OP 27 GZ op amp (not shown). The 578 op amp gain trim resistors are also tight film, ultra low drift, thin film, built-in resistors.

The control programs, the control electronics and the assembly project are designed in such a way that portability of the well-to-well and instrument-to-instrument PCR protocols is possible.

High-throughput laboratories benefit from instruments that are easy to use by a broad spectrum of laboratory personnel and that require a minimum amount of training. The program of the invention was developed to handle complex PCR thermal cycling protocols while retaining ease of programming. In addition, it is provided with the protection that ensures sample integrity during power interruptions, and can document in detail the events of each execution in a secure memory.

After completing the startup self-tests shown in Figures 53 and 54, to assure the operator that the system is working properly, the user interface of the invention offers a simple, high-level menu that invites the user to execute, create or edit a file, or access a utility function. No programming skills are required, as pre-existing default files with custom times and temperatures can be quickly edited and then stored in memory for later use. A file protection scheme prevents unauthorized changes to any user program. A file typically consists of a series of instructions for maintaining a desired temperature or for thermal cycling. Complex programs are created by linking files together to form a method. A commonly used file, such as a 4 ° C incubation followed by thermal cycling, can then be stored and incorporated into methods created by other users. A new type of file, the AUTO file is a PCR cycling program that allows the user to specify which of several types of control parameter changes will take place in each cycle: time increments (to another segment extension, to improve performance). performance), decrease in time, or increase or decrease in temperature. For the highest degree of control precision and for the most reliable method transfer, temperatures are adjustable by 0.1 ° C and times are programmed to the nearest second. The invention has the possibility of

ES 2 318 232 T3 program a scheduled PAUSE at one or more set points during a run for reagent additions or for removing tubes in specific cycles.

The system of the invention has the capacity to store a historical file of 500 records for each execution. This feature allows the user to review the individual steps in each cycle and flag any special status or error messages regarding irregularities. With the optional printer, the invention provides written documentation of the file and method parameters, run time / temperature data with a date / time stamp, configuration parameters, and classified file directories.

To ensure reproducible thermal cycling, the calculated sample temperature is indicated during the change and hold segments of each cycle. A temperature one degree lower than the set temperature is normally used to trigger the change and hold timers, although this can be altered by the user. As long as the appropriate time constant for tube type and volume is used, the sample will always approach the desired sample temperature with the same precision, regardless of whether long or short sample incubation times have been programmed. Users can program slow changes for specialized hybridization requirements of degenerate primer sets, or very short (1-5 seconds) high temperature denaturation periods for very GC rich targets. Smart default PCR cycles for 2- and 3-temperatures are pre-programmed.

Any user can access the diagnostic tests to check the status of the heating and cooling systems, as the program provides Pass / Fail reports. In addition, a system performance program performs a comprehensive evaluation of the subsystems and generates a health summary report.

The control program (firmware) is made up of several sections that are listed below:

• Diagnostics • Calibration • Installation • Real-time operating system • Nine prioritized tasks that manage the system • Boot sequence • User interface

The various sections of the program will be described either with their textual description, pseudocode or both.

The characteristics of the program (firmware) are:

1. A control system that manages the mean temperature of the sample block to within +/- 0.1 ° C while also maintaining temperature non-uniformity between wells in the sample block within +/- 0.5 ° C.

2. A temperature control system that measures and compensates for fluctuations in line voltage and temperature drift of the electronics.

3. Extensive startup diagnostics that determine if system components are working

Four. Extensive diagnostics in the installation program that check heating and cooling systems to ensure they are working properly.

5. A logical and organized user interface, employing a menu driven system that allows instrument operation with minimal dependence on the operator's manual.

6. The ability to link up to 17 PCR protocols and store them as one method.

7. The ability to store up to 150 PCR protocols and methods in the user interface.

8. A history file that records up to 500 events from the previous run as part of the sequence task.

9. The ability to define the reaction volume and tube size type at the beginning of a run for maximum precision and temperature control as part of the user interface and that modifies the tau (the tube time constant) in the PID task.

ES 2 318 232 T3

10. Upon recovery from a power failure, the system brings the sample block to 4 ° C to protect any samples that may be loaded in the sample compartment. The analyzer also reports the duration of the power failure as part of the sequence task.

eleven. The ability to print the archive content, “run” parameters, and stored PCR protocol parameters as part of the print job.

12. The ability to configure which state the device will return to during any stopped state.

13. The ability to verify that the setpoint temperature is reached within a reasonable amount of time.

14. The ability to control the instrument remotely through an RS232 port.

There are several levels of diagnosis which are described below:

A series of startup tests are performed automatically each time the instrument is turned on. Critical areas of the hardware are evaluated without user intervention. Any test that detects a component failure will be run again. If the test fails, an error message is displayed and the keyboard is electronically locked to prevent the user from continuing.

The following areas are tested:

Programmable peripheral interface device

Battery powered RAM device

RAM checksum

EPROM devices

Programmable interface timer devices

Clock / calendar device

Programmable interrupt controller device

Analog to digital section

Thermometers

Verify the proper configuration connector

A series of service-only diagnostics are available for final tests at the manufacturing site or for field service engineers via a “hidden” key sequence (ie, unknown to the customer). Many of these tests are the same as those for the startup diagnostics except that they can be run continuously up to 99 times.

The following areas are tested:

Programmable peripheral interface device

Battery powered RAM device

RAM checksum

EPROM devices

Programmable interface timer devices

Clock / calendar device

Programmable interrupt controller device

Analog to digital section

RS-232 section

ES 2 318 232 T3

Display section

Keyboard

Acoustic warning

Quick cooling valves

Checking for EPROM Mismatch

Program version level (firmware)

RAM checksum and initialization

Auto start program indicator

Clear calibration indicator

Heated deck heater and control circuits

Edge heater and control circuits

Manifold heater and control circuits

Central heater and control circuits

Sample Block Thermal Cut Test

Heated Cover Thermal Break Test

User diagnostics are also available to allow the user to perform a quick heat and cool test and extensive confirmation of the cooling and heating system. These diagnostics also allow the user to view the history file, which is a sequential record of events that occurred in the previous run. Registers contain time, temperature, set point number, cycle number, program number, and status messages.

Remote diagnostics are available to allow control of the system from an external computer through an RS-232 port. Control is limited to service diagnostics and instrument calibration only.

Calibration is performed to determine various parameters such as heater resistance, etc. Access to the calibration screen is limited by a “hidden” key sequence (ie, unknown to the customer). The following parameters are calibrated:

The configuration connector that resets the cooling unit, sample block heaters, coolant pump, and power supply for the appropriate voltage and frequencies (100 V / 50 Hz, 100 V / 60 Hz, 120 V / 60 Hz, 220 V / 50 Hz or 230 V / 50 Hz). The user enters the type of installed configuration manifold. The program uses this information to calculate the equivalent resistance of the sample block heaters. After startup, the system verifies the selected configuration connector is consistent with the current line voltage, frequency.

The heater resistance must be determined in the calibration process so that accurate calculations of the heater power delivered can be made. The user enters the actual resistances for the six sample block heaters (two main heaters, two manifold heaters, and two edge heaters). The configuration connector physically wires the heater in series for 220-230 V ac and in parallel for 100-120 V ac The program calculates the equivalent resistance of each of the three heaters with the following formula:

For 100-120 V ac: R_eq = (Ri * R₂) / R₁ + R₂

For 220-230 V ac: R_eq = R! + R₂ (7) (8)

Equivalent resistance is used to provide a precise amount of heating power to the sample block (Power = Voltage² x Resistance).

Calibration of the A / D circuit is necessary so that temperatures can be accurately measured. This is done by measuring two voltage test points (TP6 and TP7 in the CPU circuit) and entering the voltages

ES 2 318 232 T3 measurements. The A / D output for each voltage is the basis of the two calibration points on the curve. These voltages are derived from the precision 5V source and are accurate and independent of temperature. At the beginning of each run, these voltages are read by the system to measure the electronic drift due to temperature because any change in the A / D output is due to temperature dependencies in the analog chain (multiplexer, analog amplifier and A / D converter).

Calibration of all four temperature detectors (sample block, ambient, refrigerant, and heated cover) is performed for accurate temperature measurements. Before their installation in an instrument, the temperature detectors of the environment, refrigerant and heated cover are placed in a water bath where their output is recorded (XX, X ° C at YYYY mV). These values ​​are then entered into the system. A one-point calibration curve is used as the accuracy of the temperature in those areas is not critical.

The sample block detector is calibrated on the instrument. An array of 15 precision temperature probes is strategically placed in the sample block in the preferred embodiment. The output of the temperature waves is collected and averaged on a computer. The block program commands bring it to 40 ° C. After a short stabilization period the user enters the mean block temperature as read by the 15 probes. This procedure is repeated at 95 ° C, forming the two-point calibration curve.

Calibration of the AC to DC line voltage sampling circuit is performed by inputting the AC to DC circuit output into the system for two given AC input voltages, forming a two-point calibration curve. The output of the circuit is not linear in the required range (90 - 260 V ac) and therefore requires two points on each terminal (100 and 120, 220 and 240 V ac), but only uses one set based on the input voltage current.

An accurate measurement of AC voltage is necessary to deliver a precise amount of power to the sample block (Power = Voltage² x Resistance).

The installation program is a diagnostic test that performs a comprehensive test of the heating and cooling systems. The installation measures or calculates the conductance of the control cooling, the conductance of the rapid cooling at 10 ° C and 18 ° C, the cooling power at 10 ° C and 20 ° C, the heating and cooling capacity of the control block. samples and the delay in the sample block detector. The purpose of the installation is threefold:

1. Uncover faulty or marginal components.

2. Use some of the measured values ​​as system constants stored in battery-powered RAM to optimize the control system for a given instrument.

3. Measure cooling system degradation and heating over time.

The installation runs once before shipping the system and should also be run before use or when any major component is replaced. The installation program can also be run by the user under the user diagnostics.

The heater boost test verifies that the heaters are properly configured for the current line voltage (ie, in parallel for 90-132 V ac and in series for 208-264 V ac). The program supplies a power pulse to the sample block and then monitors the rise in temperature within a period of 10 seconds. If the temperature rise is outside of a specified range for the rate of rise, then the heaters are incorrectly wired for the current line voltage and the installation process is terminated.

Control refrigeration conductance tests measure thermal conductance K_DC through the sample block to the control refrigeration steps. This test is performed by first bringing the temperature of the sample block to 60 ° C (the fast valves are closed), and then integrating the power of the heater required to keep the block at 60 ° C in a period of time of 30 seconds. The integrated power is divided by the sum of the difference between the block and coolant temperatures during the interval.

(9) K_DC = Σ Heater Power₆o ° c / Σ Block Temp - Refrig Temp

Typical values ​​are 1.40 to 1.55 W / ° C. A low Kcc may indicate a clogged line (s). A high Kcc can be due to a fast valve that is not fully closed, refrigerant leaks to the outer diameter of the sleeve, or a sleeve that has dislodged.

The Block Thermal Capacity (Cap Blq) test measures the thermal capacity of the sample block by first controlling the block at 35 ° C and then applying maximum power to the heaters for 20 seconds. The thermal capacity of the block is equal to the integrated power divided by the difference in the temperature of the block. To increase precision, the effect of the control cooling power is subtracted from the integrated power.

ES 2 318 232 T3 (10) Cap Blq = ramp time * (heater power - control cooling) / delta temp where:

<td>ramp time =</td><td>20 seconds</td>

<td>heater power =</td><td>500 watts</td>

<td>refrig. control =</td><td>(Σ Block Temp - Refrig Temp) * K_DC</td>

<td>delta temp =</td><td>TBblock_{t = 2}or - TBloque_t=₀</td>

The typical value of Cap Blq is 540 watt-seconds / ° C ± 30. Assuming a value of K_DC Normally, an increase in the thermal capacity of the block is due to an increase in thermal loads, such as moisture in the cover foam, loss of insulation around the sample block, or a decrease in heater power such as a failure in one of the six heater zones, or a fault in the electronic circuit that operates the heater zones, or an incorrect or incorrectly wired voltage configuration module.

A cooling test measures the cooling output of the system in watts at 10 ° C and 18 ° C. The system's cooling power, or cooling output, at a given temperature is equal to the sum of thermal loads at that temperature. The main components are: 1. heating power required to keep the block at a given temperature, 2. power dissipated by the pump used to circulate the refrigerant around the system, and 3. losses in the refrigerant lines to the environment. The cooling power parameter is measured by controlling the coolant temperature at both 10 ° C and 18 ° C and integrating the power applied to the sample block to maintain a constant coolant temperature, for an interval of 32 seconds. The difference between the temperature of the coolant and the block is also integrated to calculate the losses towards room temperature.

(11) Cooling pot = Σ Heating power + Pump power + (Kamb * Σ (block temp - cooling temp) where:

<td>Heating power =</td><td>Sum of the heating power required to keep the refrigerant at 10 ° C or 18 ° C for 32 seconds</td>

<td>Pump power =</td><td>Circulation pump, 12 watt</td>

<td>Kamb =</td><td>Ambient conductance, 20 watts / ° C</td>

<td>temp block - temp refrig =</td><td>sum of the differences in block and coolant temperature during the 32 seconds.</td>

Typical value for cooling power is 230 watts ± 40 at 10 ° C and 370 watts ± 40 at 18 ° C. Low cooling power may be due to a clogged fan duct, a faulty fan, or a failing or marginal cooling unit. It may also be due to a poorly wired voltage configuration connector.

The blast chill conductance tests measure the Kc thermal conductance at 10 ° C and 18 ° C through the sample block to the blast chill steps. This test is performed by first monitoring the coolant temperature at 10 ° C or 18 ° C, and then integrating, over a 30 second interval, the applied heater power to maintain the coolant at the given temperature divided by the block and the coolant in the time interval.

(12) K_c = Σ Heater Power / Σ (Block Temp - Refrigeration Temp)

ES 2 318 232 T3

Typical values ​​for K_c they are 28 watts / ° C ± 3 at 10 ° C and 31 watts / ° C ± 3 at 18 ° C. A low K_c This may be due to a closed or clogged speed valve, kinked coolant lines, weak pump, or a hard water / Prestone (antifreeze) mixture.

A detector delay test measures the delay of the block detector by first monitoring the temperature of the block at 35 ° C and then applying 500 watts of heating power for 2 seconds and measuring the time required for the block to rise 1 ° C. Typical values ​​are 13 to 16 units, where each unit equals 200 ms. A slower or longer detector delay may be due to a poor interface between the detector and the block, such as a lack of thermal paste, a poorly made detector cavity, or a defect in the detector.

The remaining installation tests are currently run by the installation program but have a limited diagnostic purpose due to the fact that they are calculated values ​​or are a function of so many variables that their results do not accurately determine the source of a problem.

The installation program calculates the slope of the fast cooling conductance (S_c) between 18 ° C and 10 ° C. It is a measure of the linearity of the conductance curve. It is also used to approximate the ramping cooling conductance at 0 ° C. Typical values ​​are 0.40 ± 0.2. The spread of the values ​​confirms the fact that it is only an approximation.

<img file="ES2318232T3_D0003.tif" />

The installation program also calculates the cooling conductance K_c0. K_c0 it is an approximation to the conductance of the refrigeration at 0 ° C. The value is extrapolated from the actual conductance at 10 ° C. Typical values ​​are 23 watts / ° C ± 5. The formula used is:

<img file="ES2318232T3_D0004.tif" />

The installation program also calculates the cooling capacity (Cap Refr) which is an approximation to the thermal capacity of the complete refrigerant chain (refrigerant, piping lines, heat exchanger and valves). The cooling capacity is equal to the components that pump heat into the refrigerant minus the components that remove heat from the refrigerant. The mechanism used to measure and calculate these components is complex and is described in detail in the description section of the source code. To this extent, the refrigerant is allowed to stabilize at 10 ° C. Maximum heating power is applied to the sample block for a period of 128 seconds.

(15) Cap Laughing = Heat Sources - Cooling Sources (16) Cap Laughing = Pot. Heater + Pot. Bomba + Kamb * (ETamb ZTrefrig) - Cap Blq * (TBIoque_{t = 0} - TBIoque_t= i28) - Average cooling power between Trefrig_t= oy Trefrig_t= i28

The characters enclosed in {} indicate the variable names used in the source code.

Heater Impulse Test Pseudocode:

The heater impulse test verifies that the heaters are properly wired for the current line voltage.

Bring the sample block and the coolant to a known and stable point.

Turn ON the rapid cooling valves

Wait for block and coolant to get below 5 ° C

Disconnect (OFF) the rapid cooling valves

ES 2 318 232 T3

Measure the cooling effect of the cooling control by measuring the block temperature drop over a 10 second interval. Wait 10 seconds for stabilization before taking any measurements.

Wait 10 seconds templ = block temperature

Wait 10 seconds temp2 = block temperature {tempa} = temp2 - templ

Examine the variable (linevolts) that contains the actual measured line voltage. Pulse the heater with 75 watts for a line voltage greater than 190 V or with 300 watts if it is less than 140 V.

if ({linevolts}&gt; 190 volts) then deliver 75 W to heater else deliver 300 W to heater

Measure the temperature rise over a period of 10 seconds. The result is the average heat index at 0.01 ° / second.

templ = block temperature

Wait 10 seconds temp2 = block temperature {tempb} = temp2 - templ

Subtract the mean heat index {tempb} from the effect of the cooling control to calculate the true heating index (17) heat index = {tempb} - {tempa}

Evaluate the Index_heat. For 220V-230V, the heating rate should be less than 0.30 ° / second. For 100V-120V the heating rate should be greater than 0.30 ° / second.

if (line voltage = 220V and heating_index&gt; 0.30 ° / second) then

Error -&gt; Heaters wired for 120

Lock keyboard if (line voltage = 120V and heating_index &lt;0.30 ° / second) then

Error -&gt; Heaters wired for 220

Lock keyboard

K-test pseudocode_DC:

This test measures the conductance of the control refrigeration also known as K_DC.

K_DC it is measured at a block temperature of 60 ° C.

Bring the block to 60 ° C

Keep the temperature of the block at 60 ° C for 300 seconds

ES 2 318 232 T3

Integrate the power being applied to the sample block heaters during the 30 second period. Measure and integrate the power required to maintain the temperature of the block with the control cooling.

(dt_sum) = O (sum of delta temperatures) {main_pwr_sum} = O (sum of main heater power) (aux_pwr_sum) = 0 (sum of auxiliary heater power) for (count = 1 to 30) {

{dt_sum} = {dt_sum} + (block temperature - coolant temperature)

Wait 1 second

Accumulate the power applied to the main and auxiliary heaters. The actual code resides in the PID control task and is therefore summed every 200 ms.

{main_pwr_sum} = {main_pwr_sum} + (actual_power) {aux_pwr_sum} = {aux_pwr_sum} + (auxl_actual) + (aux2_actual)}

Calculate the conductance by dividing the sum of the power by the sum of the temperature. Note that the units are 10 mW / ° C.

<img file="ES2318232T3_D0005.tif" />

Pseudocode of the CP BLOCK test:

This test measures the thermal capacity of the sample block.

Bring the block to 35 ° C

Control the temperature of the block at 35 ° C for five seconds and record the initial temperature.

temperature = block temperature to give maximum power to the heaters for 20 seconds while adding the temperature difference in the block to the coolant, as well as the power of the heater.

Give 500 watts {dt_sum} = 0 for (count = 1 to 20 seconds) {

{dt_sum} = (dt_sum) + (block temperature - coolant temperature)

Wait 1 second}

ES 2 318 232 T3 (19) delta_temp = block temperature - initial temperature

Calculate the joules in cooling power due to the control cooling that takes place during the ramp.

(20) refrigjoules = Control refrigeration conductance (K_DC) * {dt_sum}

Calculate the total joules applied to the block by the main heater and control cooling. Divide by the temperature changes during the interval to calculate the thermal capacity.

(21) CP Block = ramp time * (refrigerant heater power) / deltatemp where:

ramp time = 20 seconds heater power = 500 watts

COOL_PWR_10:

This test measures the cooling power at 10 ° C.

Control the coolant temperature at 10 ° C and stabilize it for 120 seconds.

count = 120 do while (count! = 0) {

if (refrigeration temperature = 10 ± 0.5 ° C) then count = count - 1 else, count = 120

Wait 1 second}

At this point, the coolant has been at 10 ° C for 120 seconds and has stabilized. Integrate, for 32 seconds, the power applied to maintain the coolant temperature at 10 ° C.

ES 2 318 232 T3 {cool_init} = coolant temperature {main_pwr_sum} = O (aux_pwr_sum) = O {delta_temp_sum} = O for (count = 1 to 32) {

Accumulate the power applied to the main and auxiliary heaters. The actual code resides in the control task.

{main_pwr_sum} = {main_pwr_sum} + actual_power (aux_pwr_sum) = (aux_pwr_sum) + auxl_real + aux2_real delta_temp_sum = delta_temp_sum + (ambient temp - refrig temp)

Wait 1 second}

Calculate the number of joules of energy added to the mass of the refrigerant during the integration interval. "(Coolant_temp - cool_init)" is the change in cooling temperature during the integration interval. 550 is the capacity of the refrigerant in joules, thus the product is in joules. Represents the extra heat added to the refrigerant that causes it to deviate from the set point during the integration interval. This error is then subtracted from the total heat applied before calculating the cooling power.

(22) cooljnit = (refrigerant temp - cooljnit) * 550 J

Add the sum of the main power to the sum of the auxiliary heater to obtain the joules dissipated in 32 seconds. Divide by 32 to get the mean joules / second.

(23) {mainpwrsum} cooljnit) / 32 ({mainpwrsum} + {auxpwrsum}

Calculate the cooling power at 10 ° C by adding all the components of the cooling power.

(24) Power = {main_pwr_sum} + POT_BOMB + (K_AMB * delta_temp_sum) where:

{main_pwr_sum} =

POT_BOMB = delta_temp_sum =

K_AMB = sum of the power of the heater in the range watts, pump circulating the refrigerant sum of amb - refrigerant during the range watts / K, thermal conductance of cooling to the environment.

KC_10 test pseudocode:

This test measures the conductance of blast chilling at 10 ° C.

Control the coolant temperature at 10 ° C ± 0.5 and allow it to stabilize for 10 seconds.

ES 2 318 232 T3

At this point, the refrigerant is at its set point and is being monitored. Integrate, during an interval of 30 seconds, the power that is being applied to the heaters to keep the coolant at 10 ° C. Add the difference between the block and coolant temperatures.

{main_pwr__sum} = 0 {aux_pwr_sum} = 0 (dt_sum) = 0 for (count = 1 to 30) {

Accumulate the power applied to the main and auxiliary heaters. The actual code resides in the PID control task.

{mainpwrsum} = {main_pwr_sum} + actual_power {aux_pwr_sum} = {aux_pwr_sum} + auxl_real + aux2_real (dt_sum) = {dt_sum} + (block temp - refrig temp)

Wait 1 second}

Calculate the energy in joules delivered to the block during the integration period. Units are at 0.1 watts.

<img file="ES2318232T3_D0006.tif" />

Divide the summed power by the sum of the block-coolant temperature to obtain the fast cooling conductance at 100 mW / K.

<img file="ES2318232T3_D0007.tif" />

COOL_PWR_ 18 test pseudocode:

This test measures the cooling power at 18 ° C.

Bring the sample block and coolant to a known, stable point. Control the coolant temperature at 18 ° C and stabilize it for 128 seconds.

count = 128 do while (count! = 0) {

if (refrigeration temperature = 18 ° C + 0.5) then count = count - 1 else count = 120

Wait 1 second)

ES 2 318 232 T3

At this point, the coolant has been at 18 ° C for 120 seconds and has stabilized. Integrate, for 32 seconds, the power being applied to maintain the coolant temperature at 18 ° C.

{cool_init} = coolant temperature {main_pwr_sum} = O (aux_pwr_sum) = O (delta_temp_sum) = O for (count = 1 to 32) {

Accumulate the power applied to the main and auxiliary heaters. The actual code resides in the control task.

(main_pwr_sum} = {main_pwr_sum} + actual_power (aux_pwr_sum) = (aux_pwr_sum) + auxl_real + aux2_real {delta_temp_sum} = (delta_temp_sum) + (ambient temp - refrig temp)

Wait 1 second}

Calculate the number of joules of energy added to the mass of the refrigerant during the integration interval. "(Coolant_temp - cool_init)" is the change in cooling temperature during the integration interval. 550 is the capacity of the refrigerant in joules, thus the product is in joules. Represents the extra heat added to the refrigerant that causes it to deviate from the set point during the integration interval. This error is then subtracted from the total heat applied before calculating the cooling power.

(27) cool_init = (coolant temp - cool_init) * 550

Add the sum of the main power to the sum of the auxiliary heater to obtain the joules dissipated in 32 seconds. Divide by 32 to get the mean joules / second.

(28) {main_pwr_sum} cooljnit) / 32 ({mainpwrsum} + {auxpwrsum}

Calculate the cooling power at 18 ° C by adding all the components of the cooling power.

(29) Power = {main_pwr_sum} + POT_BOMB + (K_AMB * delta_temp_sum) where:

{main_pwr_sum} =

POT_BOMB = delta_temp_sum =

K_AMB = sum of the power of the heater in the range watts, pump circulating the refrigerant sum of amb - refrigerant during the range watts / K, thermal conductance of cooling to the environment.

ES 2 318 232 T3

KC_ 18 test pseudocode:

This test measures the conductance of blast chilling at 18 ° C.

Control the coolant temperature at 18 ° C ± 0.5 and allow it to stabilize for 10 seconds.

At this point, the refrigerant is at its set point and is being monitored. Integrate, during an interval of 30 seconds, the power that is being applied to the heaters to keep the coolant at 18 ° C. Add the difference between the block and coolant temperatures.

{main_pwr_sum} = 0 {aux_pwr_sum} = 0 {dt_sum} = 0 for (count = 1 to 30) {

Accumulate the power applied to the main and auxiliary heaters. The actual code resides in the control task.

{main_pwr_sum} = {main_pwr_sum} + actual_power {aux_pwr_sum} = {aux_pwr_sum} + auxl_real + aux2_real {dt_sum) = {dt_sum} + (block temp - refrig temp)

Wait 1 second}

Calculate the energy in joules delivered to the block during the integration period. Units are at 0.1 watts.

(30) {mainpwrsum} = {mainpwrsum} + {auxpwrsum}

Divide the summed power by the sum of the block-coolant temperature to obtain the fast cooling conductance at 100 mW / K.

<img file="ES2318232T3_D0008.tif" />

SENLAG test pseudocode:

This test measures the delay in the sample block detector.

Bring the block to 35 ° C. Keep it within ± 0.2 ° C for 20 seconds and then record the temperature of the block.

{tempa} = block temperature

Give 500 W of power to the sample block.

ES 2 318 232 T3

Apply 500 W of power for the next 2 seconds and count the number of iterations through the loop so that the temperature of the block increases 1 ° C. The iteration loop runs every 200 ms, so the actual detector delay is equal to the * 200 ms count.

seconds = 0 count = 0 do while (TRUE) {

if (seconds&gt; = 2 seconds) then turn off heaters if (block temperature - temp&gt; 1, O ° C) then exit while loop count = count + 1}

end do while delay detector = count

Pseudocode of CP Refrigerant test

This test calculates the cooling capacity of the entire system.

Stabilize the coolant temperature at 10 ° C ± 0.5 ° C.

Send a message to the PID control task to quickly raise the refrigeration temperature from its current value (around 10 ° C) to 18 ° C.

Wait for the refrigerant to cross 12 ° C so that the cooling coefficient ramp always starts at the same temperature and has already clearly started to rise. Record the initial ambient and block temperatures.

do while (cooling temperature &lt;12 ° C) {

Wait 1 second}

(blk_delta) = block temperature (h2o_delta) = coolant temperature

During the next two minutes, while the coolant temperature is rising to 18 ° C, add the coolant temperature and the difference between the coolant and ambient temperatures.

{temp_sum} = 0 {cool_sum} = 0 for (count = 1 to 128 seconds) {

{cool_sum} = {cool_temp_sum} + coolant temperature {temp_sum} = ambient temp - refrig temp

Wait 1 second count = count + 1}

ES 2 318 232 T3

Calculate the change in temperatures during the two minute period.

(34) {blk_delta} = block temperature - {blk delta} (35) {h2o_delta} = coolant temperature - {h2o_delta}

Calculate KChill, that is, the rate of change of the cooling power with the temperature of the coolant in the cooling range of 10 ° C to 20 ° C. Note that the units are in watts / 10 ° C.

(36) KChill = (Cooling pot @ 18 ° C - Cooling pot @ 10 ° C)

Calculate Sc which is the slope of the rapid cooling conductivity versus the temperature range of 18 ° C to 10 ° C. Units are in watts / 10 ° C / 10 ° C.

<img file="ES2318232T3_D0009.tif" />

Calculate Kc_0, the rapid cooling conductance extrapolated to 0 ° C.

<img file="ES2318232T3_D0010.tif" />

Calculate Cp_cool, the capacity of the refrigerant using:

(39) Cpcool = (POTCALORIF * 128 + POT_PUMP * 128

- Pot_0 ° C * 128

- CP_Block * blk_delta + K_AMB * temp_sum

- Kchill * cool_temp_sum) / h2o_delta where:

<td>POTCALORIF =</td><td>500 W, the heat power applied to heat the block, and hence heat the coolant. It is multiplied by 128, since the warm-up interval was 128 seconds.</td>

<td>POT_BOMB =</td><td>12 watts, the power of the pump that circulates the refrigerant multiplied by 128 seconds</td>

<td>Pot_0 ° C =</td><td>Cooling power at 0 ° C multiplied by 128 seconds</td>

<td>CP_Bloque =</td><td>Thermal capacity of the sample block.</td>

<td>blk_delta =</td><td>Change in block temperature during heating interval.</td>

<td>K_AMB =</td><td>20 watts / K, ambient cooling thermal conductance.</td>

<td>temp_sum =</td><td>The sum, once per second, of the ambient temperature - refrigerant during the interval.</td>

<td>h2o_delta =</td><td>Change in coolant temperature during warm-up interval (approximately 6 ° C).</td>

<td>KChill =</td><td>Slope of cooling power versus coolant temperature.</td>

ES 2 318 232 T3 cool_temp_sum = The sum of the coolant temperature, once per second, during the warm-up interval.

Real-time operating system - cretin

CRETIN is a standalone, multitasking kernel that provides system services to other programming modules called tasks. Tasks are written in the “C” language with some time critical areas written in the Intel 8085 assembly language. Each task has a priority level and provides an independent function. CRETIN resides in low memory and runs after the boot diagnostics have been run successfully.

CRETIN manages task scheduling and only allows one task to run at a time. CRETIN receives all hardware interrupts thus enabling tasks to wait to execute when the appropriate interrupt is received. CRETIN provides a real-time clock to allow tasks to wait for timed events or to stop during known intervals. CRETIN also provides communication between tasks through system message nodes.

The program (firmware) consists of nine tasks, which are briefly described below in order of their priority. Later sections will describe each task in more detail.

1. The control task (PID) is responsible for controlling the temperature of the sample block.

2. The keyboard task is responsible for processing keyboard inputs from the keys.

3. The timer task waits for the half-second hardware interrupt, then sends a wake-up message to both the sequence and screen tasks.

Four. The sequence task runs the user programs.

5. The pause task handles keyboard and scheduled pauses when a program is running.

6. The screen task updates the screen in real time.

7. The print job handles RS-232 port communication and printing.

8. The task of the LED is responsible for activating the heating LED. It is also used to control the coolant temperature while the installation is running.

9. The links task starts files that are linked together in a method by simulating a keystroke.

Block temperature control program (PID Task)

The Proportional Integral Differential (PID) task is responsible for controlling the absolute temperature of the sample block in 0.1 ° C, as well as controlling the non-uniformity of the temperature of the sample block (TNU, Temperature Non-Uniformity, defined as the temperature of the warmest well minus the temperature of the coldest well) by less than ± 0.5 ° C by applying more heating power to the perimeter of the block to compensate for losses across the edges of the protection. The PID task is also responsible for controlling the temperature of the heated jacket with a lesser degree of precision. This task runs 5 times per second and has the highest priority.

The amount of heating or cooling power provided to the sample block is derived from the difference or “error” between the user-specified sample temperature stored in memory, called the set point, and the current sample temperature. calculated. This scheme follows standard practice in control loops. In addition to a power contribution to the film heaters that is directly proportional to the current error, that is, the proportional component, (set point temperature minus the sample block temperature), the calculated power also incorporates an integral term that serves to cancel any static error (temperature set point minus block temperature less than 0.5 ° C). This component is called the integral component. To avoid the accumulation of the integral term or "slip", the contributions of the integral are restricted to a small band around the temperature set point. The gains of the proportional and integral components have been carefully selected and tested, as the time constants associated with the block and sample tube detector severely restrict the phase margin of the system, thus creating a potential for loop instabilities. The proportional gain term is P in equation (46) below and the integral gain term is Ki in equation (48) below.

The PID task uses a "controlled bypass algorithm" whereby the temperature of the block often exceeds its final steady state value so that the sample temperature reaches its desired temperature as quickly as possible. Using the bypass algorithm causes the temperature of the block to be exceeded significantly

ES 2 318 232 T3 controlled but does not cause the sample temperature to be exceeded. This saves power and is believed to be new to PCR instrumentation.

The total power delivered to all the heaters in the sample block to obtain the desired lift rate is given by:

(40) Power = (CP / ramp_index) + control where:

CP = Thermal mass of the adjustment block = power of the control cooling or control ramp_index = T_end - T_initial/ desired elevation index

This power is limited for safety to a maximum of 500 W of heating power.

With each iteration of the task (every 200 ms) the system applies the heating or rapid cooling power (if necessary) based on the following algorithms.

The control system is governed by the calculated sample temperature. Sample temperature is defined as the mean temperature of the liquid in a thin-walled plastic sample tube placed in one of the wells of the sample block (hereinafter the "block"). The time constant of the system (sample tube and its contents) is a function of tube type and volume. At the beginning of a run, the user enters the tube type and the amount of the reaction volume. The system calculates a resulting time constant (τ or tau). For the “MicroAmp” tube and 100 microliter reaction volume, tau is approximately 9 seconds.

(41) (42)

Tblock-new- ^— Tblock Power * (200mS / CP)

Tsample-new- ”Tsample + (Tblock-new- Tsample) * 200ms / tau where:

T_block-new- = current block temperature

T_block = temperature of the block makes 200 ms power = power applied to the block

CP = thermal mass of the block _T^TO sample-new current sample temperature

T = shows sample temperature 200 ms ago

<td>tau =</td><td>thermal time constant of the sample tube, adjusted for the delay in the detector (approximately 1.5)</td>

The error in the temperature signal is simply:

(43) error = Set point - T_deadstra-new As in any closed-loop system, a corrective action (heating or cooling power) is applied to cancel part of the current error. In equation (45) below, F is the fraction of the error signal to be canceled in a sample period (200 ms).

(44) Tsample-new- ⁼ Tsample + F * (PA - Tsample)

ES 2 318 232 T3 where:

PA =

User temperature set point

Due to the large delay in the system (large tube time constant), the fraction F is set small.

Combining formulas (42) and (44) we obtain:

Gour-new- ^— Tsample + (Tblock-new- ”Tsample) * 0.2 / tau Tsample + F * (PA - Tsample)

Combining formulas (41) and (45) and adding the P term (the gain of the proportional term) to limit the oscillations of the block temperature and improve the stability of the system, we obtain:

(46) Power = CP * P / T * ((SP - T_sample) * F * tau / T + T_samplestra

<img file="ES2318232T3_D0011.tif" />

where:

P = the gain of the proportional term and

T = the sample period of 0.2 seconds (200 ms).

P / T = in the preferred embodiment

Equation (46) is a theoretical equation that gives the power (power) necessary to move the temperature of the block to some desired value without taking into account the losses to the environment through the protection bands, etc.

Once the power required to change the block is determined by means of equation (46), this power is divided into the power to be delivered to each of the three zones of the heater, dividing by the areas of these zones. The losses to the collectors are then determined and a power term that is of sufficient magnitude to compensate for these losses is added to the amount of power to be delivered to the collector heater zone. In the same way, another term of power sufficient to compensate for the power lost by the block clamping bolts, the block temperature sensor and the environment is added to the power to be delivered to the edge heater zone. These additional terms and dividing the power by the area of ​​the zones converts equation (46) into equations (3), (4), and (5) given above.

Equation (46) is the formula used by the preferred embodiment of the control system to determine the heating or cooling power required for the sample block.

When the calculated sample temperature is within the “integral band”, that is, ± 0.5 ° C around the target temperature (PA), the gain of the proportional term is too small to cancel the remaining error. Therefore an integral term is added to the proportional term to cancel out small errors. The integral term is disabled outside the integral band to prevent the accumulation of a large error signal. The algorithm within the "integral band" is as follows:

(47) lnt_sum (new) = lnt_sum (old) + (PA - T_mUestra) (48) Pot_add = Ki * lnt_sum (new) where:

Int_sum = sum in the sample period of the difference between the set point (PA) and the temperature ^Tsample, ^Y

Ki = the integral gain (512) in the preferred embodiment.

ES 2 318 232 T3

Once the heating power has been calculated, the control program distributes the power to the three zones of the film heaters 254, 262, and 256 of FIG. 13 based on the area in the preferred embodiment. Edge heaters receive additional power based on the difference between the block temperature and the ambient temperature. Similarly, manifold heaters receive additional power based on the difference between the block temperature and the coolant temperature.

Pseudocode of the PID

At system startup or reset

Disconnect fast cooling

Disconnect all heaters

Calculate heater resistances

Do always - runs every 200 ms

If (block temperature&gt; 105) then

Disconnect heaters

Open fast valves

Show error message

Read line voltage (linevolts)

Read refrigerant detector and convert to temperature {h2otemp}

Read the ambient detector and convert to temperature {ambtemp}

Read detector from heated cover and convert to temperature {cvrtemp}

Read the detector from the sample block and convert to temperature {blktemp}.

That part of the code also reads the temperature stable voltage reference and compares the voltage with the reference voltage that was determined during instrument calibration. If there are any discrepancies, the electronics have drifted and the voltage readings from the temperature detectors are adjusted accordingly to obtain accurate temperature readings.

Calculate the sample temperature {tubetenths} ​​or the displayed temperature using a digital low-pass filter.

<img file="ES2318232T3_D0012.tif" />

where:

TT_neither = last sample temperature (tubetenths)

TB_n = current block detector temperature {blktenths}

T = sample interval in seconds -200 ms tau = tube tau {cf_tau} - detector tau {cf_lag}

Equation (49) represents the first term of a Taylor series expansion of the exponential that defines the calculated sample temperature given above as equation (6).

ES 2 318 232 T3

Calculate the temperature of the foam underneath the sample block, (phantenths) known as the phantom mass. The temperature of the phantom mass is used to adjust the power delivered to the block by taking into account the heat flow to and from the phantom mass. The temperature is calculated by means of a digital low-pass filter implemented in the program.

(50) phantenths = TT_n.i + (TB_n - TT_n-i) * T / taUf_Oam where:

TT_n-1 = last ghost mass temperature {phantenths}

TB_n = current block detector temperature {blktenths}

T = sample interval in seconds -200 ms tau_foam = foam block tau - 30 calculate sample temperature error (difference between sample temperature and set point temperature) {abs_tbe_err}.

Determine the direction of change (fast_ramp) = UP_RAMP or DN_RAMP

If (sample temperature is within ERR set point (PA)) then

PID not in fast transition mode (fast_ramp) = OFF where

ERR = the temperature width of the “integral band”, that is, the error band surrounding the target or set point temperature.

Calculate the current control cooling power {cool_ctrl} to determine how much heat is being lost in the control cooling channels.

Calculate the current fast cooling power {cool_ramp}

Calculate {cool_brkpt} · {cool_hrkpt} is the cooling power used to determine when to transition from fast to control cooling on descending ramps. It is a function of the block and coolant temperature.

The control cooling power {cool_ctrl} and the fast cooling power {cool_ramp} are all factors that the CPU must know to control the descending temperature ramps, that is, to calculate how long to keep the cooling solenoid actuated valves open. fast. The control cooling power is equal to a constant plus the temperature of the coolant times the thermal conductivity from the block to the control cooling channels. Likewise, the blast cooling power is equal to the difference between the block temperature and the coolant temperature multiplied by the thermal conductivity from the block to the blast chilling channels. The cooling cut-off point is equal to a constant multiplied by the temperature difference between the block and the coolant.

Calculate the heating or cooling power {int_pwr} needed to change the temperature of the block from its current temperature to the desired temperature set point (PA).

<img file="ES2318232T3_D0013.tif" />

where:

XP at proportional gain = P / T in equation (46) = approximately one in the preferred embodiment

CP = thermal mass of the block

PA = temperature set point

ES 2 318 232 T3

T_sample = sample temperature

T_block = block temperature cf_kd = Tau * K_d/ delta_t where tau is the same tau used in equation (49) and K_d is a constant and delta_t is the 200 ms sample period.

If (sample temperature is within (cf_band) of set point) then

Integrate the sample error {i_sum} else (52) clear {i_sum = 0}

Calculate the integral term of the power.

(53) integral term = {i_sum} * constant * {cf_term}

Add the integral term to the power.

(54) {int_pwr} = {int_pwr} + integral term

Adjust the power to compensate for the heating load due to the effects of the phantom (foam coating) by first finding the power of the phantom mass and then adding it to the power {int_pwr}.

Calculate the power of the phantom mass {phan_pwr} using:

(55) phant_pwr = C * (blktenths - phantenths) / 10

Where: C = thermal mass of the foam coating (1.0 W / K)

Set heater power {int_pwr} = (int_pwr) + (phan_pwr)

Calculate the power needed in the manifold heaters {aux1_pwr} which will compensate for the losses from the sample block to the edges of manifolds that have refrigerant circulating through them. Note that if the system is on a descending ramp {aux_pwr} = 0. The power required by the collector zone is described below:

(57) {aux1_pwr} = K1 * (Troque Tamb) + K2 * (Tbloque ~ Trefrig) + K5 * (dT / dt) where:

<td>K1 =</td><td>coefficient (cf_1coeff)</td>

<td>K2 =</td><td>coefficient (cf_2coeff)</td>

<td>K5 =</td><td>coefficient {cf_5coeff}</td>

<td>dT / dt =</td><td>exchange ratio</td>

ES 2 318 232 T3

<td>^Tblock -</td><td>block temperature</td>

<td>^Tamb =</td><td>room temperature</td>

<td>T = J-refrig</td><td>coolant temperature</td>

calculate the power needed in the edge heaters {aux2_power} that compensates for the losses from the edges of the sample block to the environment. Note that if the system is on a descending ramp {aux2_power} = 0. The power required by the edge zone is described below:

(58) {aux2_power} ~ K3 * (Tblock “Tamb) K4 * (Tblock Trefrig) K6 (dT / dt) where:

<td>K3 =</td><td>coefficient {cf_3coeff}</td>

<td>K4 =</td><td>coefficient {cf_4coeff}</td>

<td>K6 =</td><td>coefficient {cf_6coeff}</td>

<td>dT / dt =</td><td>exchange ratio</td>

<td>^Tblock ⁼</td><td>block temperature</td>

<td>^Tamb =</td><td>room temperature</td>

<td>T = refrig</td><td>coolant temperature</td>

Remove the power contribution to the manifold heaters {aux1_power} and edge {aux2_power} to have the total power that should be supplied to the main heaters and coolants.

(59) {int_pwr} = {int_pwr} - {aux1_power} - {aux2_power}

Decide if rapid cooling should be applied. Note that {cool_brkpt} is used as a switch point from fast cooling to control cooling.

If (int_pwr &lt;cool_brkpt and ramping down) to decide that if the block temperature is much higher than the set point temperature, rapid cooling is needed then

Open fast else valves

Close fast valves and rely on control cooling

At this point, {int_pwr} contains the total heater power and {aux1_power} and {aux2_power} contain the losses from the block to the edges. The power supplied to the auxiliary heaters is made up of two components: aux_power and int_power. Power is distributed {int_pwr} to main and auxiliary heaters based on area.

ES 2 318 232 T3 total_pwr = int_pwr int_pwr = total_pwr * 66% aux1_power = total_pwr * 20% + auxl power aux2_power = total_pwr * 14% + aux2_power

Calculate the number of half cycles that the triac 5 has to drive for each end zone and each iteration of the control loop to send the appropriate amount of power to the heaters. This loop runs once every 1/5 of a second, so there are 120/5 = 24 half cycles at 60 Hz or 100/5 = 20 at 50 Hz. The number of half cycles is a function of the required power {int_pwr }, the current line voltage {linevolts} and the heater resistance. Since the exact power required may not be delivered to each block, a remainder {delta_power} is calculated to keep track of what to include from the previous loop.

<img file="ES2318232T3_D0014.tif" />

Calculate the number of 1/2 cycles to keep the triac connected. The index is equal to the number of cycles to keep the triac on.

(61) index = wattage * ohms of main heaters *

[20 or 24] / {linevolts}²

Where equation (61) is performed once for each heater zone and where "power" = int_pwr for the main heater zone, aux1_pwr for the manifold heater zone, and aux2_pwr for the edge heater zone.

Calculate the amount of actual power delivered.

(62) actual_power = {linevolts} squared * main heater index / resistance

Calculate the remainder to be added next time.

(63) delta_power = {int_pwr} - real power

Calculate the number of 1/2 cycles for the edge and collector heaters using the same technique described for the main heaters by substituting {aux1_pwr} and {aux2_pwr} in equation (60).

Load the calculated counts into the counters that control the main, collector, and edge triacs.

Observe the heated cover detector. If the heated deck is below 100 ° C, then charge the heated deck counter to deliver 50 watts of power.

Observe the temperature of the sample. If it is greater than 50 ° C, turn on the HOT LED to warn the user not to touch the block.

End loop always

Keyboard Task

The purpose of the keyboard task is to wait for the user to press a keyboard key, compare the key with a list of valid keystrokes for the current state, execute the command function associated with the valid key, and switch to a new one. condition. Invalid keystrokes are indicated by an audible warning and ignored. This task is the heart of the state-triggered user interface. It is “state-triggered” because the action taken depends on the current state of the user interface.

ES 2 318 232 T3

Keyboard task pseudo code

Initialize the variables of the keyboard task.

Turn off the cursor.

If (install flag not set) then

Run setup program.

Send a message to the PID task to connect the deck heater.

If (found the feed when the user was running a program) then

Calculate and display the number of minutes the power was off.

Write a power failure status record to the history file.

Send a message to the sequence task to start a stabilization at 4 ° C.

Give the user the option to review the archive.

If (user needs to review the archive) then

Go to show the historical file

Show the top level screen.

Do always

Send a message to the system that this task is waiting for a hardware interrupt from the keyboard.

Go to sleep until this interruption is received.

When you woke up, read and encode the key on the keyboard.

By having a list of valid keys for the current state.

Compare the key with the list of valid keys.

If (the key is valid for this state) then

Get the action on next state information for this key

Run the action (a command function) for this state.

Go to the next state.

else

Give an acoustic warning of invalid key.

End Loop Always

ES 2 318 232 T3

Timer task overview

The purpose of the timer task is to activate the sequence task and the real time display every half second. The timer task asks the system (CRETIN) to wake up when it receives the half-second hardware interrupt that is generated by the clock / calendar device. The timer task then successively sends 2 messages to the sequence task and the real time display task respectively. This intermediate task is necessary since CRETIN will only serve one task per interrupt and hence only the task with the highest priority (the sequence task) would be executed.

Timer task pseudocode

Do always

Send a message to the system that this task is waiting for a hardware interrupt from the clock / calendar device.

Go to sleep until this interruption is received.

When you are awake, send a message to the sequence task and the real-time display task.

End Loop Always

Sequence task overview

The purpose of the sequence task is to execute the content of a user-defined program. Sequentially passes through each set point in a cycle, consisting of a ramp segment and a maintenance segment, and sends the set point temperature messages to the PID task which in turn controls the temperature of the sample block . At the end of each segment, it sends a message to the real-time display task to change the display and a message to the printer task to print the execution information for the segment. The user can stop a running program by pressing the PAUSE key on the keyboard and resume the program by pressing the START key. The user can prematurely abort a program by pressing the STOP key. This task runs every half second when it is awakened by the timer task.

(Program goes to next page)

ES 2 318 232 T3

Sequence Task Pseudocode

Do always

Initialize the variables of the sequence task.

Wait for a message from the keyboard task that the user has pressed the START key or selected START from the menu or a message from the link task that the next program in a method is ready to run.

Go to sleep until this message is received.

When you wake up, update the ADC calibration readings to account for any drift in the analog circuitry.

If (stabilization sequence not started at 4 ° C due to power failure) then

Send a message to the printer task to print the PE title line, system date and time, program configuration parameters, program type and its number.

If (started a MAINTENANCE program) then

Get the temperature to hold {hold_tp}.

Get the number of seconds to hold {hold_time}.

If (falling more than 3 ° C and (hold_tp)&gt; 45 ° C) then Send intermediate set point message.

Else

Send final set point message {hold_tp}. While (discounting the hold time {hold_time})

Wait half a second for the timer task message.

Check if the block detector is open or shorted. If (keyboard task detected PAUSE key) then

Send the current sample temperature set point.

Send a message to wake up the pause task.

ES 2 318 232 T3

Go to sleep until awakened by the pause task. Send the pre-pause set point.

If (an intermediate set point was sent) then

Send the final set point.

If (the set point temperature is below room temperature and will be there for more than 4 minutes) then

Activate a check mark to tell the PID task to connect the heated cover.

Increase the keep time counter by half seconds (store_time).

Sending the final set point again in case the hold time has expired before the intermediate set point has been reached - this ensures that the correct set point will be written to the history file.

Write a data record to the archive.

Send a message to the printer job to print the MAINTENANCE information.

End MAINTENANCE program

Else If (starting a CYCLE program) then

Add the total number of seconds in a cycle {secs_in_run}, taking into account the ramp time of the instrument and the ramp maintenance times programmed by the user.

Obtain the total number of seconds in the program by multiplying the number of seconds in a cycle by the number of cycles in a program {num_cyc}.

Total {secs_in_run} = {secs_in_run} per cycle * {num_cyc}

While (discounting the number of cycles {num_cyc})

While (discounting the number of set points (num_sec))

Get the ramp time {ramp_time}.

Obtain the final set point {t_final}.

Get hold time {local_time}.

ES 2 318 232 T3

Send a message to the real-time display task to display the ramp segment information.

If (the user programmed a ramp time) then Calculate the error (ramp_err) between the programmed ramp time and the actual ramp time as follows. This equation is based on empirical data.

(ramp_err) = prog ramp_rate * 15 + 0.5 (ramp up) (ramp_err) = prog ramp_rate * 6 + 1.0 (ramp down) where:

prog ramp_rate = (abs (T_F - T_to) - 1) / {ramp_time} T_F = set temperature {t_final} T_to = current block temperature (blktemp) abs = absolute value of expression

Note: the -1 is there because the clock starts at 1 ° C from the set point.

new ramp_time = old {ramp_time} (ramp_err)

If (new ramp_time&gt; old {ramp_time}) then new ramp_time = old {ramp_time}.

Else new ramp_time = 0.

While (the sample temperature is not within the setpoint temperature set by the user (cf_clk_dev)) Wait for wake up message from the timer task.

Send a new ramp set point every second.

Else If (ramp down by more than 3 ° C and {t_final}&gt; 45 ° C) then

ES 2 318 232 T3

Send an intermediate set point

While (the sample temperature is not within the setpoint temperature set by the user {cf_clk_dev})

Wait for wake up message from timer task.

Increase the ramp time counter by half a second.

Check if the block detector is open or shorted.

If (keyboard task detected a PAUSE key) then

Send a set point of the current sample temperature.

Send message to wake up pause task

Send the pre-pause set point.

Send the final set point.

While (the sample temperature is not within the setpoint temperature set by the user (cf_clk_dev))

Wait for wake up message from timer task.

Increase the ramp time counter by half a second.

Check if the block detector is open or shorted.

If (keyboard task detected a PAUSE key) then

Send a set point of the current sample temperature.

Send message to wake up pause task

Send the set point prior to the

ES 2 318 232 T3 pause.

Send a message to the printer task to print the ramp information.

Give an acoustic warning to signal the end of the ramp segment.

Send a message to the real-time display task to display the ramp segment information.

While (discounting maintenance time)

Wait half a second for the timer task message.

Increase the maintenance time counter by half a second.

Check if the block detector is open or shorted.

If (keyboard task detected PAUSE key) then

Send the set point as the current sample temperature.

Send a message to wake up the pause task.

Go to sleep until awakened by the pause task.

Send the pre-pause set point. Write a data record to the archive. Send a message to the printer job to print the maintenance information.

If (the final set point has deviated more than the user configurable amount {cf_temp_dev}) then Write an error log to the archive. Check if there is a scheduled pause.

Go to the next segment.

Send a message to the printer task to print an end of cycle message.

Go to the next cycle.

End CYCLE program.

ES 2 318 232 T3

Else If (starting an AUTO-CYCLE program) then

Add the total number of seconds in each program {secs_in_run} taking into account the ramp time of the instrument and the maintenance times programmed by the user that can be automatically increased or decreased by a programmed amount each cycle.

While (discounting the number of cycles {num_cyc})

While (discounting the number of set points {num_seg})

Obtain the final setpoint temperature {t_final}. Get hold time (time_hold} Check if the user programmed an automatic increase or decrease of the temperature set point and / or hold time and adjust accordingly.

If (automatic temperature increase or decrease causes the set point to fall below 0 ° C or above 99.9 ° C) then

An error log is written to the archive.

The set point is trimmed to either 0 ° C or 99.9 ° C.

Send a message to the real-time display task to display the ramp segment information.

If (ramp down by more than 3 ° C and {t_final}&gt; 45 ° C) then Send an intermediate set point

While (the sample temperature is not within the setpoint temperature set by the user {cf_clk_dev})

Wait for wake up message from timer task.

Increase the ramp time counter by half a second.

Check if the block detector is open or shorted.

ES 2 318 232 T3

If (keyboard task detected a PAUSE key) then

Send a set point of the current sample temperature.

Send message to wake up pause task. Go to sleep until awakened by the pause task.

Send the pre-pause set point.

Send the final set point.

While (the sample temperature is not within the setpoint temperature set by the user {cf_clk_dev})

Wait for wake up message from timer task.

Increase the ramp time counter by half a second.

Check if the block detector is open or shorted.

If (keyboard task detected a PAUSE key) then

Send a set point of the current sample temperature.

Send message to wake up pause task. Go to sleep until awakened by the pause task.

Send the pre-pause set point.

Send a message to the printer task to print the ramp information.

Give an acoustic warning to signal the end of the ramp segment.

Send a message to the real-time display task to display the ramp segment information.

While (discounting maintenance time)

Wait half a second for the timer task message.

ES 2 318 232 T3

Increase the maintenance time counter by half a second.

Check if the block detector is open or shorted.

If (keyboard task detected PAUSE key) then

Send the current sample temperature set point.

Send a message to wake up the pause task.

Go to sleep until awakened by the pause task.

Send the pre-pause set point. Write a data record to the archive. Send a message to the printer task to print maintenance information.

If (the final set point has deviated more than the user configurable amount (cf_temp_dev)) then Write an error log to the archive.

Go to the next segment.

Send a message to the printer task to print an end of cycle message.

Go to the next cycle.

End AUTO-CYCLE program.

Else If (starting a POWER FAILURE sequence) then Send a 4 ° C set point.

Set a {subamb_hold} flag such that the PID task will disconnect the heated cover.

Do always

Wait for a wake-up message from the half-second timer task.

Increase the maintenance time counter by half a second.

End Loop Always

End Power Failure Sequence

ES 2 318 232 T3

Write an end of execution status record to the archive.

If (executing a method)

Set a {weird_flag} flag so that the bindings task will know how to send a message to the sequence task to start the execution of the next program.

Else

Return the user interface to the idle state screen.

End Loop Always

Pause task overview

The purpose of the pause task is to handle either a pause that the user schedules in a CYCLE program or a pause when the user presses the PAUSE key on the keyboard.

When the sequence task encounters a scheduled pause in the execution of a CYCLE program, it goes to sleep and wakes up to the pause task. The pause task in turn sends a message to the real-time display task to continually display and decrease the time the user requested as a pause. When the snooze timer expires, the snooze task sends a message to wake up the sequence task and then it goes to sleep. The user can prematurely resume the program by pressing the START key on the keyboard or can prematurely abort the program by pressing the STOP key.

When the keyboard task detects a PAUSE key during program execution, it sets a flag (pause_flag) and then waits for the sequence task to recognize it. When the sequence task sees this pinned flag, it sends an acknowledgment message back to the keyboard task which then puts itself to sleep. When the keyboard task receives this message, it wakes up to the pause task. The pause task sends a message to the real-time display task to continuously display and increase the amount of time the program is paused. The timer will end when it reaches the pause time limit set by the user in the configuration section. The user can resume the program by pressing the START key on the keyboard or abort the program using the STOP key.

(Program goes to next page)

ES 2 318 232 T3

Pause task pseudo code

Do always

Wait for a message from the keyboard task indicating a keyboard pause, or a message from the sequence task indicating a user-programmed pause.

Go to sleep until a message is received.

When awakened, check the mark of the type of pause initiated.

If (it's a scheduled pause) then

Send a message to the real-time viewer task to show the pause timer counting up.

Else

Send a message to the real-time viewer task to show the pause timer counting down.

While (counting down the time counter)

Send a message to the system to suspend this task for half a second.

Send a message to the printer job to print the pause information.

If (it's a scheduled pause) then

The pause has timed out so send a message to wake up the sequence task.

Send a message to the live view task to stop the pause viewer.

Send a message to the real-time display task to resume the running program screen.

Else (it's a keyboard pause)

The pause has expired and the program must be aborted, therefore send a message to the system to stop the sequence task and send it back to the

ES 2 318 232 T3 start of your ALWAYS loop.

If (the running program was a

MAINTENANCE) then

Send a message to the printer job when printing maintenance information.

Write a status record to the archive.

Return the user interface to its idle state.

Show an aborted message.

End Loop ALWAYS.

View task overview

The purpose of the real-time display task is to display temperatures, timers, detector readings, ADC channel readings, and other parameters that need to be continuously updated every half second.

Display task pseudo code

Initialize the variables of the visualization task.

Do always

Wait for a message every half second from the timer task.

Go to sleep until a message is received.

When awakened, check if another task has sent a list of parameters to display or a flag to stop the update in progress.

Toggle the half-second mark (halt_sec)

If (there is a list of parameters to display) then

Set a traffic light so that no one else will update the screen.

Turn off the cursor.

While (passing through the parameter list)

If (is a time parameter) then

Visualize the time.

If (the half-second mark {halt_sec} is set) then

ES 2 318 232 T3

Increase or decrease the time variable.

Else if (is a decimal number) then Display a decimal number.

Else if (is an integer) then

Display the integer.

Else if (it's a read from an ADC channel) then

Read the ADC channel accounts.

If (needs to be displayed as mV) then

Convert the counts to mV.

Visualize the value.

Else if (is the power display) then Display the power in terms of watts.

Else if (is the hours remaining parameter) then Convert seconds to decimal hours. Show the hours remaining in decimal hours. If (the half-second mark {halt_sec} is set) then Decrement the seconds variable.

If (cursor was active) then

Make it active again.

Store current system time in battery powered RAM.

Clear the traffic light to release the screen.

End Loop ALWAYS

Print job overview

The purpose of the print job is to handle printing during execution. It is a low priority task and should not interfere with other time critical tasks.

Print task pseudo code

Do always

Wait for a message from another job that you want to print.

Go to sleep until a message is received.

ES 2 318 232 T3

When awakened, make local copies of the global variables to be printed.

Send a printer acknowledgment message.

If (a status or error message needs to be printed) then Print the information contained in the current history record.

Else if (print header required) then Print company name, instrument ID, program version number (firmware), and current system time and date.

Else if (you need to print the program configuration parameters) then

Print tube type, reaction volume, and sample temperature deviation from the set point that starts the clock.

Else if (end of cycle information needs to be printed) then

Print the end time and temperature.

Else if (you need to print the segment information) then Print the segment information either ramp or maintenance.

Else if (a pause status message needs to be printed) then Print the amount of time paused and at what temperature.

End Loop Always

LED task overview

The purpose of the LED task is to control the illumination of the “heating” LED by reflecting the power applied to the main heater. It is a low priority task that runs once per second.

LED task pseudo code

Initialize the variables of the LED task.

Do always

Send a message to the system to wake up this task every second.

ES 2 318 232 T3

Go to sleep.

When you wake up, load PIC timer counter 2

A with a value that reflects the power applied to the main heater as follows:

Load counter with value = {K_htled} * (ht_led)

Where:

(K_htled) contains a constant to calculate the time that the heating LED pulse equals 15200 / 500. 15200 is somewhat higher than the PIC clock or 14.4 kHz and this is the value loaded into the timer to maintain the LED constantly on. 500 is the power of the main heater.

(ht_led) will be a value between 0 and 500 and will be equal to the watts applied to the main heater.

End Loop Always

Link task overview

The purpose of the link task is to simulate that the user presses the START key on the keyboard. This task is necessary so that the programs can run one after the other (as in a method) without user intervention. The link task wakes up the sequence task and begins execution of the next program as if the START key had been pressed.

Link task pseudo code

Initialize the variables of the binding task.

Do always

If (the {weird_flag} flag is active and is not the first file in the method) then

Send a message to the sequence task to wake it up.

End Loop Always

Boot sequence

Power-on sequence

When the power is connected to the instrument or the program performs a RESET, the following sequence takes place. Note: The numbers below correspond to the numbers in the flow chart.

1. Transmits a Ctrl-G character (decimal 7) to the RS-232 printer port. It interrogates the RS-232 port for at least one second and if a Ctrl-G is received, it is assumed that an external computer is connected to the port and that all communication during the power-up sequence will be redirected from the keyboard to the RS-232 port . If Ctrl-G is not received, the power-up sequence continues normally.

2. Check if the MORE key is pressed. If so, go directly to hardware diagnostics for service only.

ES 2 318 232 T3

3. The following three tests are audio / visual checks and cannot announce an error: 1) the buzzer sounds 2) the hot, cool, and warm keyboard LEDs are flashing 3) every pixel on the screen is lit. The copyright and instrument identification screens are displayed while the power-up diagnostics are running.

Four. If an error occurs in one of the power-up diagnostics, the name of the failed component is displayed and the keypad locks except for the code “MORE 999” which will give access to the hardware diagnostics for service only.

5. Check channel zero of the PPI-B device to see if the automated test bit is low. If so, run the UART test. If the test passes, sound the buzzer continuously.

6. Start the CRETIN operating system which in turn will dock each task by priority level.

7. Check a mark on the battery powered RAM to see if the instrument has been calibrated. If not, display an error message and lock the keypad except for the code "MORE 999" which will give access to the hardware diagnostics for service only.

8. Run a test that measures line voltage and frequency and see if both values ​​conform to the selected configuration connector while calibrating the instrument. If not, display an error message and lock the keypad except for the code "MORE 999" which will give access to the hardware diagnostics for service only.

9. Perform the heater boost test as described in the installation section. If the heaters are incorrectly wired, display an error message and lock the keypad except for the "MORE 999" code which will give access to the hardware diagnostics for service only.

10. Check a mark on the battery powered RAM to see if the instrument has been installed. If not, display an error message and lock the keypad except for the code "MORE 999" which will give access to the hardware diagnostics for service only.

eleven. If not in remote mode, check a mark on battery powered RAM to see if there was a power failure while the instrument was running. If so, start a stabilization at 4 ° C and display the amount of time the power was off. Ask the user if they want to see the history file which will tell them exactly how far along the execution was when the power was lost. If you select yes, go directly to user diagnostics.

12. Sound the buzzer and erase the mark remotely so that all communication is now via the keyboard.

13. Check a mark on battery powered RAM to see if the manufacturer wants the test program to start automatically. If so, start program execution and reset the instrument after it is executed.

14. Show the top-level user interface screen.

Referring to Figure 50, there is shown a sectional view of a larger volume, thin-walled reaction tube marketed under the trademark MAXIAMP. This tube is useful for PCR reactions where it is necessary to add reagents or other materials to the reaction mix which will take the total volume to be greater than 200 microliters. This larger tube is shown in figure 50 made with Himont PD701 polypropylene or Valtec HH-444 polypropylene and has a delegate wall in contact with the sample block. Whatever material is selected, it should be compatible with the DNA and other components of the PCR reaction mix so that it does not impede the PCR reaction process such as having the target DNA adhering to the walls and not replicating. Glass in general is not a good choice because DNA has been known to stick to the walls of glass tubes.

Dimension A of Figure 50 is typically 0.3 ± 0.025mm (0.012 ± 0.001 inch) and the angle of the wall relative to the longitudinal axis of the tube is typically 17 °. The advantage of a 17 ° wall angle is that while a downward force produces good thermal contact with the sample block, the tubes do not get stuck in the sample wells. The advantage of the thin walls is that they minimize the delay between changes in the temperature of the sample block and the corresponding changes in the temperature of the reaction mixture. This means that if the user wants a reaction mixture to remain within a range of 1 ° C to 94 ° C for 5 seconds in the denaturation segment, and programs these parameters, he or she achieves denaturation in 5 seconds with less delay. longer than conventional thick-walled tubes. This product feature of being able to program a short stabilization interval such as a denaturation stabilization of 5 seconds and obtain a stabilization at the programmed temperature for the exact programmed time is made possible by the use of a calculated sample temperature to control the timer. In the system described here, the

ES 2 318 232 T3 timer that times an incubation or stabilization interval is not started until the calculated sample temperature reaches the programmed stabilization temperature.

Additionally, with thin-walled sample tubes, getting the sample to within 1 ° C of the target temperature only takes half to two-thirds of the time compared to prior art thick-walled microcentrifuge tubes and this it is true for both the tall MAXIAMP ™ tube shown in figure 50 and the smaller thin-walled MCROAMP tubes shown in figure 15.

The wall thickness of both MAXIAMP ™ and MICROAMP ™ tubes is closely controlled in the manufacturing process to be as thin as possible consistently with adequate structural strength. Typically, for polypropylene, this will be somewhere between 0.009 to 0.012 inches (0.23 to 0.3 mm). If new, more exotic materials are used that are stronger than polypropylene to gain the advantage of speeding up the PCR reaction, the wall thickness can be less as long as adequate strength is maintained to withstand the downward force that ensures good contact. thermal, and other stresses of normal use. With a height (dimension B in figure 50) of 2.85 cm (1.12 inches) and a dimension C of 1.98 cm (0.780 inches) and a wall thickness of the top section (dimension of D) of 1 cm (0.395 in), the MAXIAMP tube time constant is approximately 14 seconds although this has not been accurately measured at the time of writing. The MICROAMP tube time constant for the shorter tube shown in Figure 15 is typically about 9.5 seconds with a tube wall thickness in the tapered section of 0.23 ± 0.025 mm (0.009 ± 0.001 inches). .

Figure 51 shows the results of using a thinner-walled MICROAMP tube. A similar acceleration will be obtained reaching the target temperatures with the use of a thin-walled MAXIAMP tube.

Referring to Figure 51, a graph is shown of the relative times for the calculated sample temperature in a MICROAMP tube versus the time for a prior art tube to reach a temperature within 1 ° C of the target denaturation temperature of 94 ° C from an initial temperature of 72 ° C. In Figure 51, a 100 microliter sample was present in each tube. The curve with the data points marked by open boxes is the calculated sample temperature response for a MICROAMP tube with a response time of 9.5 seconds and a wall thickness of 0.023 cm (0.009 inches). The curve with data points marked by X represents the sample temperature calculated for a 100 microliter sample in a prior art thick walled microcentrifuge tube with a wall thickness of 0.76 mm (0.030 inches). This graph shows that the MICROAMP thin-walled sample tube reaches a temperature within 1 ° C of the 94 ° C target stabilization temperature within approximately 36 seconds while the prior art tubes require approximately 73 seconds. This is important because in instruments that do not start their timers until the stabilization temperature has basically been reached, prior art tubes can significantly increase the total processing time especially when considered in light of the fact that each PCR cycle will have at least two ramps and stabilizations and there are generally many more cycles performed. Doubling the ramp time for each ramp with the use of prior art tubes can therefore dramatically increase the processing time. In systems that start their timings based on the block / bath / furnace temperature without regard to the actual sample temperature, these long delays between changes in the block / bath / furnace temperature and the corresponding changes in the Sample mixing can have serious negative consequences. The problem is that the long delay can shorten the time that the reaction mixture is actually at the set temperature for stabilization. In very short stabilizations as are common in late PCR runs, the reaction mix may never actually reach the programmed stabilization temperature before the heating / cooling system begins trying to change the temperature of the reaction mix.

Figure 50 shows a polypropylene cap 650 connected to the MAXIAMP sample tube by plastic net 652. The outside diameter E of the cap and the inside diameter F of the top section of the tube are sized for a setting between 0, 05 and 0.13 mm (0.002 and 0.005 inches). The inner surface 654 of the tube should be free of marks, gouges and scratches so that a seal can be formed with the gas cap.

Figure 52 shows a plan view of tube 651, cap 650, and net 652. A flange 656 prevents the cap from being pushed too deeply into the tube and allows sufficient projection of the cap over the top edge of the tube. samples, to make contact with the heated pressure plate. This also allows sufficient cap deformation so that the minimum acceptable force F of Figure 15 can be applied by cap deformation.

In the preferred embodiment, the tube and cap are made of Himont PD701 polypropylene which is suitable for autoclaves at temperatures up to 126 ° C for times up to 15 minutes. This allows the disposable tubes to be sterilized before use. Because the caps permanently deform when used on machines with heated pressure plates, the tubes are designed for one-time use only.

Caps for MICROAMP tubes are available in connected strips of 8 or 12 caps with each cap numbered or as individual caps. Simple rows of caps can be used and the rows can easily be shortened to as few as desired or individual caps can be cut from the strip. The caps for the MAXIAMP tubes are either glued as shown in figure 50, or they are separate individual caps.

ES 2 318 232 T3

The maximum volume for addition of post-PCR reagents to allow mixing in a MICROAMP tube is 200 microliters and is up to 500 microliters for the MAXIAMP tube. The temperature limits are -70 ° C to 126 ° C.

The response in time depends on the volume of the sample. The response is measured as the time for the sample to reach 37% of the new temperature when the temperature of the block suddenly changes. Typical response time for a 50 microliter fill is 7.0 seconds and for a 20 microliter fill is 5.0 seconds.

Appendix a

User interface

The goal of the GeneAmp PCR System 9600 user interface is to provide a simple way to develop and run programs that perform PCR.

There are 3 types of programs available. The HOLD program consists of a single set point held for a fixed amount of time or held for an infinite amount of time and terminated by the STOP key. The CYCLE program adds the characteristics of ramps with measured time and programmable pauses. This program allows up to nine set points and up to 99 cycles. The AUTO program allows the user to increase or decrease the set point time and / or temperature a fixed amount each cycle. This program also allows up to nine set points and up to 99 cycles. A METHOD program (method) provides a way to link up to 17 “hold”, “cycle” or “auto” programs together.

A total of 150 programs numbered 1 to 150 can be stored. Programs can be created, stored, protected, printed or deleted. A directory of stored programs can be viewed or printed.

(Program goes to next page)

ES 2 318 232 T3

System 9600 Keypad

<img file="ES2318232T3_D0015.tif" />

RUN Starts execution of a program from the program (run) screen or resumes a programmed pause or keyboard.

MORE Switches between run screens and also accesses (more) functions for service only (if code 999 is followed).

BACK Moves to the previous field within the same screen. If you are currently (back) positioned in the first field, it moves to the previous screen.

STEP Moves down to the first field on the next (forward) screen.

PAUSE Starts a countdown paused by manual interruptions, (pause)

OPTION Either move the cursor left-to-right through the (option) menu items (rotating over the leftmost option) or toggle a YES / NO answer.

STOP Aborts a running program or takes the user up one level (stop) in the user interface.

CE Clears invalid numeric entries.

ENTER Accepts the current numeric entry, accepts a menu (enter) item, accepts a YES / NO answer, or moves to the next field on a screen. If the numeric entry is the last of a⁶ screen, ENTER moves to the next screen.

ES 2 318 232 T3

System 9600 Common Screens

PROGRAM screen

Example:

<td>Prog ### Msg</td><td>Temp</td>

<td>Menu</td><td></td>

CYCL # 17 Donate 74.0C

RUN-STORE-PRINT-HOME

Prog tt tt tt

Msg Temp Menu

EXECUTION screen is any of HOLD, CYCL, AUTO or METH is the program no. (1-150) or ??? if it has not been stored yet it is either Done, Error, Abort or blank is the current sample temperature are the available options

Example:

Action

Timer

Temp

Prog / Cyc

Ramp to 94.0C 29.6C

10:00 Cycle 14

Action

Temp Timer

Prog / Cyc is either 'Hold at xx.xC' (hold at xx, x ° C) or 'Ramp to xx.xC' (ramp to xx, x ° C) is the current sample temperature countdown time maintenance or ramp or count-up of FOREVER time (always).

for a HOLD file it is 'Prog xxx' for a CYCL or AUTO file it is 'Cycle xx' - count up

MORE screen (more)

Example:

Setpt

Timer

Tot Cyc

Prog

Setpt # 3 Tot Cyc 25

Hrs left 2.5 Prog 17

Setpt Tot Cyc Timer

Prog is the number of the current set point (1-9) - count up is the total number of cycles (1-99) in the current program is the remaining time of the program in hours - count down is the number of current program (1-150)

ES 2 318 232 T3

Keyboard PAUSE screen

Example:

Prog ### Temp

PAUSE timer

CAR # 18 55.OC

PAUSE 9:45

<td>Prog tt tt tt ft ff fl 1! II II Temp Timer</td><td>is either HOLD, CYCL, AUTO or METH Is it the program no. (1-150) or ??? if it hasn't been stored yet is the current sample temperature is the configurable pause time - countdown</td>

Upper level of the User Interface

Select Option 9600

RUN-CREATE-EDIT-UTIL

TOP LEVEL screen

<td>Run Enter program #xxx</td><td>Create program HOLD-CYCL-AUTO-METH</td>

<td>RUN screen</td><td>CREATE screen</td>

<td>Edit</td><td>Select function</td>

<td>Enter program #xxx</td><td>DIR-CONFIG-DIAG-DEL</td>

<td>EDIT screen</td><td>UTILITIES screen</td>

Programs are created by selecting a program type on the CREATE screen. The user is taken directly to the first editing screen of the program.

Stored programs are recalled by entering a number from 1 to 150 from the RUN, EDIT, or program screens. Entering a valid program number on the RUN screen automatically starts execution. Entering a valid program number in the EDIT or program screen takes the user to the first screen of the program edit.

ES 2 318 232 T3

Programs are edited by pressing the STEP key (moves down one screen), BACK (moves to the previous field), or ENTER (moves to the next field).

Programs are run by selecting RUN from the RUN-STOREPRINT-HOME menu or by pressing the RUN key on the keyboard. The user must first enter the two parameters required for each run.

Tube type: MICRO

React vol: lOOuL

Select print mode

OFF-CYCLE-SETPOINT

Select print mode

OFF-ON

Cover temp is xx ° C

Run starts at 100 ° C

The OPTION key changes the tube type from MICRO (MicroAmp tube) to THIN (GeneAmp thin-walled tube). If the user configured a special tube, then the OTHER option is added. A different ratio volume can be entered. These parameters are stored with this program. ENTER accepts these values. If the user set the running printer to ON and is running a cycle, auto, or method program, then the following print options are offered.

If the user set the running printer to ON and is running a maintenance program, then the following printing options are offered. If the heated cover is below 100 ° C, the following screen is displayed. If the user is on this screen when the heated cover reaches 100 ° C, it automatically starts the run. If the user presses STOP to return to the program screen, then execution must be manually resumed.

Accepting HOME on the RUN-STORE-PRINT-HOME menu without saving a program displays the screen:

ES 2 318 232 T3

Prog #xxx not stored

Continued? AND IT IS ·

Maintenance program

HOLD #xxx xx. xC

RUN-STORE-PRINT-HOME

PROGRAM screen

HOLD at xx.xC

Hold FOREVER-xxx: xx

Beep while Hold? DO NOT

The user can choose between infinite stabilization or limited time maintenance.

The buzzer will sound once per second.

MAINTENANCE SCHEDULE - Execution screens

HOLD at xx.xC xx.xC xxx: xx Prog xx

EXECUTION screen

None

MORE screen (more)

HOLD #xx xx.xC

PAUSE xx: xx

None

KEYBOARD PAUSE screen

PROGRAMMED PAUSE

MAINTENANCE SCHEDULE - Printing in progress

ES 2 318 232 T3

PE Cetus GeneAmp PCR System 9600 Ver xx.x Nov 14, 1990 xx: xx am

Tube type: MICRO Reaction vol: 100uL Start dock within x.xC of setpt

HOLD program #xxx

HOLD Program: xx.xC xxx: xx Actual: xx.xC xxx: xx or

HOLD Program: xx.xC FOREVER Current: xx.xC xxx: xx

HOLD program #xxx - Run complete Nov 14, 1990 xx: xx am

CYCLE program

CYCL #xxx xx.xC

RUN-STORE-PRINT-HOME

PROGRAM screen x Temperature PCR

Setpt # 1 Ramp xx: xx xx.xxC Hold xx: xx

The default is 3. This determines the number of set points in this program. 1 to 9 set points are allowed.

The number of setpoints previously entered determines how many setpoint edit screens will be offered.

The user can enter a ramp and hold time for each set point. The hold timer will start when the sample temperature reaches within the user-configurable set point temperature range.

ES 2 318 232 T3

<td> —</td><td>If the user does NOT want a pause, they are skipped</td>

<td>Total cycles = xx Pause during run? DO NOT</td><td>the next 3 screens. 1 to 99 cycles are allowed.</td>

Enter a 0 in the number of the point of

<td>Pause after setpt #x Beep while pause? YES</td><td>set point also means that the user does NOT want a pause, therefore the</td>

next 2 screens.

The cycle number is limited to the number

<td>lst pause at cycl xx Pause every xx cycle</td><td>total cycles previously entered.</td>

The default pause time is fixed at

<td>Pause time xx: xx</td><td>user settings.</td>

CYCLE PROGRAM - Run screens

<td colspan="2">Ramp at xx.xC xx.xC</td><td rowspan="2">Setpt #x Tot Cyc xx Hrs left XX Progxxx</td>

<td>xxx: xx</td><td>Cycle xx</td>

<td colspan="2">RUN screen (ramp) Hold at xx.xC xx.xC xxx: xx Cycle xx</td><td>MORE screen (more)</td>

RUN screen (maintenance)

ES 2 318 232 T3

CYCL #xxx xx. xC xx. xC

PAUSE xx: xx

Setpt #x xx.xC

Hrs left XX Progxxx

KEYBOARD PAUSE screen

PROGRAMMED PAUSE

CYCLE PROGRAM - Printing in progress

PE Cetus GeneAmp PCR System 9600 Ver xx.x Nov 14, 1990 xx: xx am

Tube type: MICRO Reaction vol: 100uL Start dock within x.xC of setpt

CYCL program #xxx

Cycle #xx

Setpt #x RAMP Program: xx.xC xx: xx xx: xx

HOLD Program: xx.xC xx: xx

Current: xx.xC

Current: xx.xC xx: xx (up to 9 set points) (up to 99 cycles)

CYCL program #xxx - Run complete Nov 14, 1990 xx: xx am CYCL program #xxx - User Aborted Nov 14, 1990 xx: xx am (only if aborted)

AUTO program

AUTO #xxx xx. xC

RUN-STORE-PRINT-HOME

PROGRAM screen

ES 2 318 232 T3 x Temperature PCR

Setpt # 1 xx.xC

Hold for xx: xx

<td colspan="3">Setpt # 1 xx.xC Change time / temp? YES</td>

<td colspan="3"></td>

<td>xx. xC</td><td>delta -</td><td>x. xC</td>

<td></td><td>delta</td><td>xx: xx</td>

<td colspan="3"></td>

<td>Total</td><td>cycles -</td><td>XX</td>

<td colspan="2">AUTO PROGRAM</td><td>- Screen</td>

<td colspan="2">Hold at xx.xC</td><td>xx. xC</td>

<td>xxx: xx</td><td colspan="2">Cycle xx</td>

The default is 3. This determines the number of set points in this program. 1 to 9 set points are allowed.

The number of setpoints previously entered determines how many setpoint edit screens will be offered. No ramp time is offered so the instrument will ramp as fast as possible. The hold timer will begin when the sample temperature reaches within the user-configurable set point temperature range.

If the user wishes to increase or increase the time and / or temperature each cycle, then the following screen is offered.

The OPTION key changes the arrow up (increase in each cycle) or down (decrease in each cycle). The maximum time allowed to decrease is limited to the time of holding the set point.

Up to 99 cycles are allowed.

of execution

Setpt #x Tot Cyc xx

Hrs left XX Progxxx

EXECUTION screen

MORE screen (more)

ES 2 318 232 T3

AUTO #xxx xx.xC

PAUSE xx: xx

None

KEYBOARD PAUSE screen

PROGRAMMED PAUSE

AUTO PROGRAM - Printing in progress

PE Cetus GeneAmp PCR System 9600 Ver xx.x Nov 14, 1990 xx: xx am

Tube type: MICRO Reaction vol: 100uL Start dock within x.xC of setpt

AUTO program #xxx

Hey #xx

Setpt #x RAMP Program: xx.xC xx: xx Actual: xx.xC xx: xx

HOLD Program: xx.xC xx: xx Current: xx.xC xx: xx. (up to 9 set points). (up to 99 cycles)

AUTO program #xxx - Run complete Nov 12, 1990 xx: xx am

AUTO program #xxx - User Aborted Nov 12, 1990 xx: xx am (only if aborted)

METHOD program

METH #xxx xx.xC

RUN-STORE-PRINT-HOME

RUN screen

ES 2 318 232 T3

Up to 17 can be linked in one method

<td>Link progs: - -</td><td>programs. If the user tries to enter a # of non-existent program, the</td>

message Prog does not exist. If the user tries to link another method, the message Cannot link a

<td> _____</td><td>method (Cannot bind a method.)</td>

METHOD program - Execution screens

The RUN, MORE and PAUSE screens will be those of the currently running program. Two additional MORE screens are offered when the running program is bound in a method.

............... - The number of the currently running meth #xxx aaa-bbb- program will blink.

ccc-ddd-eee-fff-ggg Additional screen MORE (more) hhh-iii-jjj-kkk-111mnun-nnn-ooo-ppp-qqq PROGRAM METHOD - Printing in progress

ES 2 318 232 T3

PE Cetus GeneAmp PCR System 9600 Ver xx.x Nov 14, 1990 xx: xx am

Tube type: MICRO Reaction vol: 100uL Start dock within x.xC of setpt method program #xxx - precedes all linked program data

METHOD program #xxx - Meth Complete - follow all linked program data

METHOD PROGRAM - Printing in progress

Select option

METHOD-PROGRAM DATA

METHOD prints the header of each program linked in the method.

PROGRAM DATA prints the header and content of each program linked in the method.

Saving a program

When STORE is selected from the RUN-STORE-PRINT-HOME menu, the routine for storing a program is the same for a file as it is for a method. Protecting a program assures the user that the program will not be overwritten or deleted without knowing the user number. Other users will be able to view, edit, run or link the protected file in their methods but they will not be able to alter the stored version.

- - ------- xxx is the first available store program number from 1 to 150.

Enter program #xxx

ES 2 318 232 T3

The user has entered the program #

<td>Progxxx is protected Enter user fxxxx</td><td>protected. The correct user # must be entered to overwrite this program.</td>

The wrong user # was entered. This

<td>Progxxx is protected Wrong user number!</td><td>screen remains for 5 seconds before returning to the previous one. User has</td>

three chances to enter the correct #.

If the user tries to overwrite a program

<td>Progxxx is linked in Methxxx Continue? YES</td><td>which is bound in a method, the user is warned and given the option to continue or not.</td>

If the user tries to overwrite a program

<td>Can't overwrite prog Linked in method xxx</td><td>which is bound in one method with another method, an error message is given.</td>

The user is given the opportunity to protect

<td>Store Protect program? DO NOT</td><td>a program as well as unprotect a previously protected program. The user wants to protect the program and therefore</td>

<td>Store Enter user txxxx</td><td>Therefore you must enter a user #.</td>

Ready to store the program in a

<td>Prog #xxx User #xxxx OK to store? AND IT IS</td><td>Available space. The user # appears only if the program is protected.</td>

Ready to overwrite a program

<td>Prog #xxx User #xxxx OK to overwrite? AND IT IS</td><td>existing. The user # appears only if the program is protected.</td>

ES 2 318 232 T3

UTILITY functions

Select function

DIR-CONFIG-DIAG-DEL

UTILITIES screen

DIR allows the user to view or print a directory of stored programs either by their program number, user number or program type.

CONFIG allows the user to tailor the use of the instrument to their specific needs.

DIAG provides the user with a means of diagnosing performance problems and verifying instrument performance.

DEL allows the user to delete stored programs either by their program number, user number or program type.

UTILITIES - DIRECTORY

Directory

PROG-TYPE-USER-PRINT

Directory

Enter program #xxx

Directory by PROGrama number.

Programs will be listed in numerical order starting at the given number. The STEP and BACK keys move through the directory screens. The buzzer sounds at the beginning or end of the list of

HOLD # 124 programs.

STOP returns the user to the previous screen.

Directory by TYPE (ΤΥΡΕ) of program.

ES 2 318 232 T3 ====================== ^^ The program numbers for the

Directory type of program selected.

HOLD-CYCL-AUTO-METH

CYCL # 15

Directory by USER number.

All programs stored under the given user number will be listed.

Directory

Enter user #xxxx

METH # 150 User # 1234

Directory PRINT

Directory Print PROG-TYPE-USER

The user can get a hard copy of the directory status in the same way that directories were viewed previously.

UTILITIES - USER CONFIGURATION

Configuration

EDIT-PRINT

Time: xx: xx

Date: mm / dd / yy

The configuration file can be edited by accepting EDIT from the menu or by pressing the STEP key. PRINT prints the content of this file.

User can set system time and date.

ES 2 318 232 T3

Runtime printer OFF

Runtime beeper ON

Pause time-out limit xx: xx

Allowed setpt error

x. x ° C

Idle state setpoint xx ° C

If Print Running is ON, the user will be prompted for print options at the beginning of each run. If the buzzer in execution is ON, then a warning will sound at the end of each segment (after the ramp or hold portion of a sequence) while executing a program.

This time represents the maximum amount of time a program can pause before it is aborted. This affects only the keyboard pause. This data represents the number of degrees that the actual sample temperature can vary from the set point before an error is signaled.

This set point is useful for balancing the control cooling power that is always present. The sample temperature will remain at this idle state set point while the instrument is idle.

The watch that times the segment of

<td>Start dock within xx ° C of setpoint</td><td>keeping a program running can be configured to be triggered</td>

when this temperature is reached the sample temperature. The nominal value is 1.0 ° C.

ES 2 318 232 T3

Special tube? DO NOT

Rxn vol = xxxuL T = xxxs

Rxn vol = xxxuL T = xxxs

If a user wants to use a different type of tube other than MicroAmp or thin-walled GeneAmp tubes, they must set this option to YES and enter at least three pairs of tube time constant and reaction volume data. This curve will be used to extrapolate the correct tau (tube time constant) for each run using this type of tube depending on the reaction volume entered by the user at the beginning of the run.

Three sets of this screen will be offered if the user sets Special tube? in YES.

UTILITIES - DELETE

Delete

PROGRAM-USER-ALL

Delete by PROGRAM

Delete

Enter program #xxx

Can't delete progxxx

Linked in methodxxx!

All programs (files and methods) can be deleted by number.

A program cannot be deleted if it is bound in a method.

Progxxx is protected

Enter user #xxxx

The user has entered the # of a protected program. The correct user # must be entered to delete this program.

ES 2 318 232 T3

Progxxx is protected

Wrong user number!

Prog #xxx User #xxxx

Delete program? AND IT IS

The wrong user # was entered. This screen remains for 5 seconds before returning to the previous one. The user has three chances to enter the correct #.

Ready to delete from the program. The user # appears only if the program was protected.

Delete by USER

Delete

Enter user #xxxx

Programs can be deleted under a given user number.

Delete

No progs with #xxxx

If there are no programs with the given user #, the following message is displayed.

Progs linked in meth

STEP to list progs

Programs cannot be deleted if they are linked in a method. The STEP key cycles through the list of linked programs.

Can't delete progxxx

Linked in methodxxx!

The list of linked programs will show in which method the program is linked.

User #xxxx

Delete all progs? YES

This will delete all programs under the user # since they are not linked.

Erase everything

Delete every unprotected prog? YES

This will delete each of the unprotected programs that are not linked in a protected method.

ES 2 318 232 T3

UTILITIES - USER DIAGNOSTICS

While running any diagnostic test, the STOP key always returns the user to the top-level diagnostic screen that automatically increments the test number and the name of the next test. This makes it easy to manually go through the available diagnostics.

Enter Diag Test # 1

REVIEW HISTORY FILE

Review historical file

Enter Diag Test # 1

REVIEW HISTORY FILE

The user can enter the number of the diagnostics to run or can use the STEP or BACK keys to go through the available tests. Each time the STEP or BACK key is pressed, the test number is increased or decreased and the name of the associated test is displayed. This feature eliminates the need for the user to memorize the number associated with each test.

The archive is a circular storage in battery-powered RAM that can store up to 500 records from the last run. When the storage is full, the oldest entries will be overwritten. The storage will be cleared automatically before the execution of a program.

ES 2 318 232 T3 ================= ^ The header of the archive file

History nnn recs

ALL-STAT-ERRORS-PRNT shows the current number of records in the file ('nnn').

ALL shows all records

STAT shows only status registers

ERRORS shows only logs with error messages

PRNT prints all or part of the archive.

The two types of registers are 1) status registers that give information about the program and 2) data registers that give information about each maintenance and ramp segment in a program. A maintenance schedule is treated as a maintenance segment and the data record will be stored when the archiving is complete.

Since there could be hundreds of inputs (50 cycles x 6 set points = 350 inputs), a fast, bi-directional movement through the file is required. Note that most PCR programs will be 3 or 6 set points and 40 cycles or less. Entries will normally be reviewed in reverse order, hence the first record viewed will be the last record written. If the user has chosen a type of record to view, STEP or BACK will take him up and down in input-to-input storage of the chosen type. Preceding STEP or BACK with a number, the second line is replaced with Skip #xxx entries. The user enters a number and presses ENTER to accept the value and that number of entries is skipped by going forward (STEP) or backward (BACK). By preceding STEP or BACK with the RUN key, the user can quickly move to the largest register # (the newest register) or to the # 1 register (the oldest register) of the chosen type.

STOP ends the review mode and displays the header of the file.

STATE REGISTRATION

ES 2 318 232 T3 .ffff # xxx / mmm nnn message

Status messages

Tube type: xxxxx

Reaction vol: xxxpL

Clk starts w / in x.xC

Start xx / xx / xx xx: xx

End xx / xx / xx xx: xx

Meth Complete

Pause xx: xx at xx.xC

Fatal status messages

Sensor error

Power fail xxx.x hrs

User Abort

Pause Timeout xx: xx 'ffff is either HOLD, CYCL or AUTO.

'xxx' is the program number

7mmm 'is the method number for a linked program, blank otherwise.

'nnn' is the record number 'message' is one of the following:

Type of sample tube used in the run.

Reaction volume used in the run. The maintenance timer starts at that set point temperature. Execution start time and date. Time and date of the end of the execution.

All programs linked in the method are complete.

The program was stopped during this time at this temperature.

One detector had bad readings 10 times in a row.

Power was off for this amount of time.

The user pressed the STOP key during execution.

The keyboard pause has reached its configurable time limit.

ES 2 318 232 T3

Fatal Setpoint Error

It is the request to abort a program if the set point has not been reached within a calculated amount of time. A 10 x 10 lookup table with the ramp start temperature (0 ° C 100 ° C in 10 ° C increments) versus the ramp end temperature (the same labels on the axis) will contain the mean time that it should take TC2 up or down a certain number of degrees. The file will be aborted if the set point is not reached in an amount of time calculated as follows: programmed ramp time + (2 * value in lookup table) + 10 minutes.

DATA LOG f # xxx / mmm ddd.dC nnn

Cycy and Setpt z look: ss' f is either HOLD, CYCL or AUTO.

'xxx' is the program number

7mmm 'is the method number for a linked program, blank otherwise.

'ddd.d' is the final set point temperature.

'nnn' is the register number 'yy' is cycle number 'z' is the set point number 'mmm: ss' is the set point time.

The cycle and set point fields will be omitted from a maintenance schedule.

DATA ERROR LOG

ES 2 318 232 T3 message ddd.dC nnn

Cycyy Setpt z look: ss' ddd.d¹ is the final set point temperature.

'nnn' is the record number 'yy' is cycle number 'z' is the set point number 'mmm: ss' is the set point time, 'message' indicates a non-fatal error as follows:

Non-fatal error messages

Setp Error

Prog Error

Temp Error

The set point was not reached in the calculated time:

programmed ramp time + (2 * value in lookup table)

An auto increase / decrease program for the temperature or set point time caused the hold time to become negative or the temperature to be outside the range 0.1 ° C to 100 ° C.

At the end of the segment, the set point temperature has drifted +/- a user configurable amount.

For the maintenance schedule, the cycle and set point fields will be omitted.

Historical archive printing

Access to the historical file printing routines is done through the header menu of the historical file. The OPTION key cyclically moves the cursor through the options:

HISTORY nnn recs

ALL-STAT-ERRORS-PRNT

Pressing the ENTER key when the cursor is positioned under PRNT displays the print screen:

100

ES 2 318 232 T3

Print History

ALL-STAT-ERRORS

ALL prints all records in the file

STAT prints only status records

ERRORS prints only records with error messages

When one of the print options is selected, the following screen is displayed:

Print History

Print from prog üxx

The first (most recent) program number will be the default program. The user can change the program number from which to start printing. While printing, the following screen is displayed:

Print History

... printing

At the end of printing, the history printing menu is displayed again.

HEATER TEST

Enter Diag Test # 2

HEATER TEST

The heater test calculates the rate of heating of the sample block when its temperature rises from 35 ° C to 65 ° C. While the block temperature is forced to 35 ° C the following screen is displayed.

Heater Test Blk = XX.X going to 35C ...

101

ES 2 318 232 T3

When the temperature stabilizes, all the heaters are turned on at full power. The display now reads going to 65C and the block temperature is monitored for 20 seconds after it passes 50C. After 20 seconds, a pass or failure message is displayed.

Heater Test PASSES

TESTING THE REFRIGERATION

Enter Diag Test # 1

CHILLER TEST

The heater test calculates the rate of cooling of the sample block when its temperature drops from 35 ° C to 15 ° C. While forcing the block temperature to 35 ° C the following screen is displayed.

Chillr Test Blk = XX.X going to 35C ...

When the temperature stabilizes, the refrigeration is switched on. The display now reads going to 15 ° C and the block temperature is monitored for 20 seconds after it passes 25 ° C. After 20 seconds, a pass or failure message is displayed.

Chiller test PASSES

102