System and method for carrying out and monitoring polymerase chain reactions

Description of equivalent WO 9746712 A2

SYSTEM AND METHOD FOR CARRYING OUT AND MONITORING BIOLOGICAL PROCESSES

The copendmg PCT application filed in the Receiving Office/United States (RO/US) on June 4, 1997 entitled Monitoring

Hybridization During PCR as serial no PCT/US/ and naming

University of Utah Research Foundation as applicant, with Carl T Wittwer, Kirk M Ririe, and Randy P Rasmussen as inventors and applicants for the United States, is hereby incorporated by reference in its entirety

BACKGROUND

1 The Field of the Invention

This invention relates generally to apparatus which are used to carry out biological processes, such as the polymerase chain reaction More specifically, the present invention relates to apparatus and methods which carry out thermal cycling and monitoring of various biological reactions, such as the polymerase chain reaction

2 The Background Art

In numerous areas of industry, technology, and research there is a need to reliably and reproducibly subject samples to thermal cycling The need to subject a sample to repeated thermal cycles is particularly acute in biotechnology applications In the biotechnology field, it is often desirable to repeatedly heat and cool small samples of materials over a short period of time One such biological process that is regularly carried out is cyclic DNA amplification

Cyclic DNA amplification, using a thermostable DNA polymerase, allows automated amplification of primer specific DNA, widely known as the "polymerase chain reaction" or "PCR " Automation of this process requires controlled and precise thermal cycling of reaction mixtures usually contained in a plurality of containers In the past, the container of preference has been a standard, plastic microfuge tube

Commercial programmable metal heat blocks have been used in the past to effect the temperature cycling of samples m microfuge tubes through the desired temperature versus time profile However, the inability to quickly and accurately adjust the temperature of the heat blocks through a large temperature range over a short time period, has rendered the use of heat block type devices undesirable as a heat control system when carrying out processes such as the polymerase chain reaction

Moreover, the microfuge tubes which are generally used have disadvantages The material of the microfuge tubes, their wall thickness, and the geometry of microfuge tubes is a hindrance to rapid heating and cooling of the sample contained therein The plastic material and the thickness of the wall of microfuge tubes act as an insulator between the sample contained therein and the surrounding medium thus hindering transfer of thermal energy Also, the geometry of the microfuge tube presents a small surface area to whatever medium is being used to transfer thermal energy The continued use of microfuge tubes in the art, with their suboptimal geometry, indicates that the benefits of improved thermal transfer (which come by increasing the surface area of a sample container for a sample of constant volume) has heretofore not been recognized Furthermore, devices using water baths with fluidic switching,

(or mechanical transfer) have also been used as a thermal cycler for the polymerase chain reaction Although water baths have been used in cycling a polymerase chain reaction mixture through a desired temperature versus time profile necessary for the reaction to take place, the high thermal mass of the water (and the low thermal conductivity of plastic microfuge tubes) , has been significantly limiting as far as performance of the apparatus and the specificity of the reaction are concerned

Devices using water baths are limited in their performance This is because the water's thermal mass significantly restricts the maximum temperature versus time gradient which can be achieved thereby Also the water bath apparatus has been found to be very cumbersome due to the size and number of water carrying hoses and external temperature controlling devices for the water Further the need for excessive periodic maintenance and inspection of the water fittings for the purpose of detecting leaks in a water bath apparatus is tedious and time consuming. Finally, it is difficult with the water bath apparatus to control the temperature in the sample tubes with the desired accuracy.

U.S Patent No 3,616,264 to Ray shows a thermal forced air apparatus for cycling air to heat or cool biological samples to a constant temperature Although the Ray device is somewhat effective in maintaining a constant temperature within an air chamber, it does not address the need for rapidly adjusting the temperature in a cyclical manner according to a temperature versus time profile such as is required for biological procedures such as the polymerase chain reaction

U S Patent No 4,420,679 to Howe and U S Patent No 4,286,456 to Sisti et al both disclose gas chromatographic ovens The devices disclosed m the Howe and Sisti et al patents are suited for carrying out gas chromatography procedures but do not provide thermal cycling which is substantially any more rapid than that provided by any of the earlier described devices Rapid thermal cycling is useful for 0 carrying out many procedures Devices such as those described in the

Howe and Sisti et al patents are not suitable for efficiently and rapidly carrying out such reactions

In particular, the polymerase chain reaction (PCR) is a fundamental DNA amplification technique essential to modern molecular 5 biology Despite its usefulness and popularity, the current understanding of PCR is not highly advanced Amplifications must be optimized by trial and error and protocols are often followed blindly The limited understanding of PCR found in the art is a good example of how those skilled in the art are content: to utilize a powerful O technique without reflection or comprehension

Biological processes such as PCR require temperature cycling of the sample. Not only does the prior art, as explained above, carry out temperature cycling slowly, the prior art also ignores the underlying principles which allow PCR to work and could be used to make PCR even more useful Thus, it would be a great advance in the art to provide methods and apparatus which are particularly adaptable for rapidly carrying out PCR and analyzing the reaction which is taking place, particularly if such reaction is analyzed as it is taking place that <img file="WO9746712A2_D0001.tif" />

BRIEF SUMMARY AND OBJECTS OF THE INVENTION In view of the above described state of the art the present invention seeks to realize the following objects and advantages

It is an object of the present invention to provide an apparatus for accurately controlling the temperature of biological samples

It is a further object of the present invention to provide a thermal cycling apparatus for quickly and accurately varying the temperature of biological samples according to a predetermined temperature versus time profile

It is another object of the present invention to provide an apparatus suitable for subjecting a number of different biological samples to rapid thermal cycling

It is also an object of the present invention to provide a thermal cycling apparatus having a thermal transfer medium of low thermal mass which can effectively subject samples to a laLge temperature gradient over a very short period of time

It is a further object of the present invention to provide an apparatus which can subject a biological sample to rapid thermal cycling using air as a thermal transfer medium

It is another object of the present invention to provide a thermal cycling apparatus which will heat samples located in a fluid chamber therein by means of an internal heater, and will subsequently cool the samples by moving ambient fluid into the chamber, at the proper time in the thermal cycle, to cool the samples

It is an object of the present invention to provide a system and method for performing PCR rapidly and for simultaneously monitoring the reaction It is another object of the present invention to provide a system and method for performing PCR rapidly and also continuously monitoring the reaction while it is ongoing.

It is a further object of the present invention to provide a ^" system and method for performing PCR rapidly while also adjusting the reaction parameters while the reaction is ongoing.

It is another object of the present invention to replace the nucleic acid probes by synthetic nucleic acid analogs or derivatives, e .g.. by peptide nucleic acids (PNA) , provided that they can also be labeled with fluorescent compounds.

These and other objects and advantages of the invention will become more fully apparent from the description and claims which follow, or may be learned by the practice of the invention.

In accordance with one aspect of the present invention, an apparatus is provided which is particularly suited for subjecting biological samples to rapid thermal cycling in order to carry out one or more of a number of procedures or processes. In one of its preferred forms, the apparatus includes a means for holding a biological sample. In some preferred embodiments, the structure which holds a biological sample, also referred to as a sample chamber, is provided with an insulation means for retaining thermal energy and also a means for heating the interior of the sample chamber. In some preferred embodiments, an incandescent lamp functions as a means for heating the interior of the sample chamber. In further embodiments, hot or cool air is conveyed into and out of a chamber holding the biological sample. In some preferred embodiments, a thermal insulator is disposed along the interior of the sample chamber and functions to retain the heat generated by the lamp within the sample chamber and serves as an insulation means. 0 In order to rapidly cool the sample chamber, the preferred apparatus includes a means for forcing air into the sample chamber and a means for dispersing the air forced into the sample chamber. The preferred structures included in some embodiments are a high velocity fan which functions to force air into the sample chamber and a rotating paddle which functions to disperse the air into the chamber In some embodiments, a means for venting allows the air to escape from the sample chamber taking the unwanted heat with it The present invention allows heating and cooling of a sample to take place both quickly and uniformly.

In accordance with the method and the apparatus of the present invention, a control structure provides means for operating the system through a desired time versus temperature profile The present invention is particularly well suited for carrying out automated polymerase chain reaction procedures

The controller of the present invention allows the biological samples to pass through a predetermined temperature cycle corresponding to the denaturation, annealing and elongation steps in the polymerase chain reaction. In use, the apparatus of the present invention allows rapid optimization of denaturation, annealing, and elongation steps in terms of time and temperature, and shortened time periods (ramp times) between the temperatures at each step

The present invention particularly decreases the total time required for completion of polymerase chain reaction cycling over prior art thermal cycling devices while at the same time significantly increasing specificity and yield

In accordance with another aspect of the present invention, the present invention provides methods and apparatus for monitoring of DNA amplification so as to track the progress of such procedures In particular, the present invention provides methods and apparatus for continuous fluorescence monitoring of the polymerase chain reaction procedure In preferred embodiments of the present invention, optical components are combined with structures to provide rapid temperature cycling in order to continuously monitor DNA amplification by a variety of different fluorescence techniques Glass capillary sample containers and composite plastic/glass sample containers allow rapid heat transfer from the preferred thermal transfer medium (allowing 30 amplification cycles in less than 15 minutes when a gas such as air is used as the thermal transfer medium) and simultaneous monitoring of the reaction.

In accordance with another aspect of the present invention, optical techniques are used to monitor the progress of the reaction as the reaction is ongoing. In some preferred embodiments of the invention, flourescent probes are added to the reaction mixture. The present invention then monitors the fluorescence at least once during a temperature transition, and preferably the fluorescence is acquired two or more times during a temperature transition, either from a single sample or from multiple samples. In some preferred embodiments a rotating carousel is included to sequentially move the samples, one-by- one, to a monitoring location with all of the samples being simultaneously subjected to rapid thermal cycling. Desirably, embodiments of the present invention provide for monitoring of fluorescence once per amplification cycle or monitoring temperature, time, and fluorescence continuously throughout each amplification cycle.

Using the present invention, a 3-dimensional plot of temperature, time, and fluorescence, can be obtained. Fluorescence vs. temperature plots of hybridization probes discriminate between the cumulative, irreversible signal of exonuclease cleavage and the temperature- dependent, reversible hybridization of adjacent probes. Hybridization probes are more useful than hydrolysis probes because the temperature dependence of fluorescence can be followed and used to detect alterations in product sequence, i.e. , polymorphisms and mutations. Using dyes that fluoresce in the presence ot double stranded DNA, product denaturation, reannealing and extension can be followed within each cycle. The present invention provides apparatus and methods for rapidly carrying out DNA amplification reactions which combines amplification and analysis of the reaction in under fifteen minutes and more preferably in under fifteen minutes and most preferably in under ten minutes . BRIEF DESCRIPTION OF THE DRAWINGS

In order to better appreciate how the above-recited and other advantages and objects of the invention are obtained, a more particular description of the invention briefly described above will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings . Understanding that these drawings depict only typical embodiments of the invention and are not therefore to be considered limiting of its scope, the invention will be described and explained with additional specificity and detail through the use of the accompanying drawings in which:

Figure 1 shows a perspective view of a thermal cycling apparatus adapted for thermal cycling of biological samples and adapted especially for use in cyclic DNA amplification, according to the concepts of the present invention

Figure 2 is a side elevation view of the fluid chamber portion of the apparatus of Figure 1

Figure 3 is an interior plan view of the fluid chamber portion of the apparatus illustrated in Figure 1

Figure 4 shows an interior plan view of the fluid chamber of another embodiment of the present invention

Figure 5 shows an optimized temperature versus time profile for a polymerase chain reaction using the thermal cycling device of the present invention

Figure 6 shows graphically the effect of denaturation time on polymerase chain reaction yields using one thermal cycling device of the present invention.

Figure 7 shows graphically the effect of annealing time on polymerase chain reaction specificity and yields using the thermal cycling device of the present invention

Figures 8A-B, which are perspective and elevational cross sectioned views, respectively, of another preferred embodiment of the present invention Figure 8C is a diagrammatic representation of the relationship of the heat producing element and the capillary tubes holding the biological samples in the embodiment illustrated in Figures 8A-B.

Figure 9A shows the results of four different temperature/time profiles (A-D) and their resultant amplification products after thirty cycles (A-D) .

Figure 9B shows a cycle of another preferred temperature/time profile used by the present invention. Figures 9C-G show exemplary cycles of other preferred temperature/time profiles used by the present invention.

Figure 10 provides a block diagram of a temperature slope control circuit in accordance with the present invention.

Figure 10A is a graphical representation of the effect of the temperature transition rate from the product denaturation temperature to the primer annealing temperature on reaction product specificity.

Figure 11 is a schematic view of a preferred rapid temperature cycler with fluorescence detection in accordance with the present invention.

Figure 11A is a temperature v. time chart of showing one preferred operation of the apparatus of Figure 11.

Figure 12 is a representation of three dimensional plots of temperature, time, and fluorescence during amplification of a hepatitis B DNA fragment in the presence of SYBR Green I .

Figures 12A-C are representations of two dimensional plots of temperature vs. time, fluorescence vs. time, and fluorescence vs. temperature which are together shown as a three dimensional plot in Figure 12.

Figure 13 is a plot of fluorescence vs . temperature during the amplification of a 536 base pair fragment of the human β-globin gene in the presence of SYBR Green I .

Figure 14 is a plot of fluorescence vs. cycle number obtained in accordance with an aspect of the present invention.

Figure 14A provides a legend for Figure 14, and subsequent figures, indicating different initial template copy numbers. Figure 15 is a plot of fluorescence vs . cycle number obtained in accordance with an aspect of the present invention.

Figure 16 is a fluorescence ratio vs. temperature plot obtained in accordance with one aspect of the present invention.

Figure 17 is a fluorescence ratio vs . temperature plot obtained in accordance with one aspect of the present invention.

Figure 18A is a graph representing an equilibrium PCR paradigm.

Figure 18B is a graph representing a kinetic PCR paradigm.

Figure 18C is a graph representing different time/temperature profiles near an annealing temperature.

Figure 19 represents another preferred embodiment of the present invention configured for continuous monitoring of a sample.

Figures 19A-19D are representations of different sample container configurations.

Figure 19E is a chart which shows the effect of the different sample container configurations of Figures 19A-D on the temperature response of the sample itself.

Figures 19F and 19G are side and end views, respectively, of one preferred sample container in accordance with the present invention.

Figures 19H and 191, respectively, show two possible orientations of a rectangular capillary tube when detecting fluorescence of the sample.

Figure 20 shows the optical layout of another preferred embodiment in accordance with the present invention to provide continuous monitoring of a sample undergoing DNA amplification.

Figure 21 is a schematic representation of another embodiment of the present invention which is a rapid temperature cycler with fluorescence detection at the tip of the sample containers .

Figures 21A-D show composite plastic/glass containers into which biological samples are loaded.

Figure 22 illustrates useful temperature vs. time segments for fluorescence hybridization monitoring.

Figure 22A charts the effectiveness of light piping by viewing the tip rather than the side of capillary sample container. Figure 22B charts the efficiency of light piping by two different sizes of capillary sample tubes.

Figure 22C is a high level block diagram showing the tasks which are performed by one preferred embodiment of the present invention which includes a rapid temperature cycler with epifluorescence detection.

Figure 22D is a plot of temperature vs time for a PCR reaction in which fluorescence feedback was used to control reaction parameters

Figure 22E is a plot of fluorescence vs time for a PCR reaction in which fluorescence feedback was used to control reaction parameters

Figure 23 is a plot of fluorescence vs time showing showing the inverse relationship between temperature and fluorescence

Figure 24 is a plot of temperature vs time showing the inverse relationship between temperature and fluorescence

Figure 25 is a plot of fluorescence vs temperature for three different PCR products m the presence of SYBR Green 1 acquired during a 0 2 degree per second temperature transition through the product melting temperatures

Figure 26 is a plot of fluorescence vs time showing product annealing for different concentrations of PCR product m the presence of SYBR Green 1.

Figures 27A and 27B are cross sectional schematic views of the embodiment represented m Figure 28 in a rjn mode and a load mode respectively

Figure 28 is a schematic representation of another embodiment of the present invention which is a rapid temperature cycler with fluorescence detection at the tip of the sample containers and which includes positioning for fluorescence detection in two dimensions to optimize detection

Figure 29 is a perspective view of the exterior of the embodiment of the present invention including the components illustrated in the schematic representation of Figure 28

Figures 30A-30V are detailed schematic diagrams of the electrical components of one preferred embodiment of the present invention Figures 31A and 31B are perspective and cross sectional views, respectively, of a sample handling system in accordance with the present invention.

Figure 32 is a schematic representation of another embodiment of the present invention which accommodates multiple sample handling trays

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Reference will now be made to the drawings wherein like structures will be provided with like reference designations

As shown in Figure 1, the one preferred thermal cycling device 10 includes a closed loop fluid (most preferably air) chamber, generally designated at 11, which is adapted to accept samples to be cycled through vent door 14 The closed loop fluid chamber 11 includes a plurality of compartments each of which will be described shortly The device 10 also includes a controller 12 which can be programmed by means of input keys 25 and display 26 to cause the chamber 11 to be cycled through a series of temperatures over a predetermined period of time The thermal cycling of chamber 11 can be used to carry out numerous procedures and is particularly suited for amplification of primer specific DNA from samples containing reaction mixtures as will be explained below.

The closed loop fluid chamber 11 is enclosed in a generallv box shaped configuration by housing 13 Blower mounting boards 16 if desired, can be located so as to section off a smaller rectangular section of the chamber 11 and function to support and secure a generally cylmdrically shaped lower housing 15 thereto Alternatively, the fan of the blower 28 may be housed integrally within chamber housing 13

The interior of blower housing 15 contains the blades and shaft of the blower The blower motor (not shown) is located externally of blower housing 15, and therefore exteriorly of the enclosed chamber 11 In this configuration, the blades and shaft are the only parts of the blower which become exposed to the circulating hot fluid within chamber 11 It would be disadvantageous to mount the motor within the chamber which would subject the motor to temperature variations and also would add the thermal mass of the motor to that which is subject to heating and cooling The reduction of thermal mass exposed to the fluid in chamber 11 is desirable to the overall performance of the device 10 in its function of subjecting samples placed therein to a desired temperature versus time profiles, using either predetermined profiles or by altering one or more reaction parameters as the reaction continues, as will be more fully explained below

The blower 28 is a well known type of blower usually identified as an "in line" type blower which preferably employs a propeller type fan, due to its generally low thermal mass, or if desired, a squirrel cage type fan, the fan preferably having a 75 cubic feet per minute minimum capacity

The solenoid platform 17 has secured thereto a solenoid 18 The solenoid armature 19 is attached to upper end 21 of rod 20 which is rigidly attached to vent door 14 and rotatably attached to housing 13 at points above and below the vent door 14 The rod 20 therefore allows vent door 14 to freely rotate relative to the housing 13 about the rod's longitudinal axis.

A spring 22 is attached at one of its ends to the housing 13 by support post 23 The opposite end of spring 22 is attached to the top end 21 of rod 20 directly adjacent the attachment of solenoid armature 19 The spring 22 is drawn between these two attachment points so as to be m tension The spring 22 therefore tends to draw top end 21 toward the support post 23, which m turn tends to rotate vent door 14 to its closed position When solenoid 18 is actuated, armature 19 tends to pull top end 21 of the rod 20 m the direction of the solenoid 18, which is opposite the direction of pull of spring 22, and which tends to open the vent door 14

Controller, generally designated at 12, is electrically attached to the chamber 11 by means of a transmission cable 24 The cable 24 also supplies power to the blower motor (not shown) , and to the heat coil 31 Further, the controller 12 also is connected to thermocouple sensor 35 for receiving signals corresponding to temperature data, and to solenoid 18 for triggering the solenoid armature

Controller 12 can be any well known type of temperature controller unit which is programmable to control the heat coil 31, vent door 14, and blower so as to achieve predetermined temperatures as a function of time within the chamber 11, and which is also capable of being programmed to actuate a relay output for driving a solenoid at predetermined time periods and chamber temperature levels A preferred temperature controller 12 for use in the embodiment of Figures 1-3 is a Partlow MIC-6000 proportional temperature controller, available through Omega Engineering Inc, of Stanford, Connecticut, as the Model No CN8600 process controller

As shown in Figures 2 and 3, the interior of chamber 11 is sectioned off into four mam compartments The blower compartment 28 is formed of the blower housing 15 and the blower mounting plates 16 The entirety of blower compartment 28 is filled with the fan and shaft portions of a blower as has been described above The blower can be any of a number of well-known designs, as has been described above, and has therefore been omitted from Figure 3 for purposes of clarity It is sufficient for the present invention to understand that the fan located in blower compartment 28 draws fluid into the blower compartment 28 through inlet opening 36 and pushes the fluid out of exit opening 37

It is preferred that the fluid be driven by the blower at a rate of at least 75 cubic feet per minute It is important however, m regard to the present invention, to realize that the fluid located in chamber 11 only contacts the fan and a portion of the drive shaft of the blower, the blower motor itself being located outside of the blower housing 15 so as to avoid any contact thereof with fluid in the chamber 11 This consideration contributes to the speed of operation of the invention to minimize the material which contacts the fluid inside the chamber 11 so as to minimize the thermal mass of material which must be heated and/or cooled thereby during the cycling process By minimizing the thermal mass which must be heated or cooled by the fluid, the response time necessary to bring the contents of chamber 11 to a uniform temperature is greatly diminished.

Fluid exiting blower compartment 28 through outlet opening 37 enters heating compartment 29. Fluid passing into heating compartment 29 must pass by heating coils 31. If the heating coils 31 get hotter than the fluid passing into heating compartment 29, the fluid will become heated thereby as it is forced through the compartment. The heating coil is preferably a 1,000 watt (125 VAC) nichrome wire coil wound around a microsupport . However, any heating unit suitable for heating the type of fluid present in the chamber may be used. The particular heating coil of embodiment of Figures 1-3 is manufactured by Johnstone Supply, of Portland, Oregon.

The heating coil is activated by an output relay included m the controller 12. The preferred relay is a 25 A, 125 VAC solid state relay manufactured by Omega Engineering Inc. of Stanford, Connecticut as Model No. Omega SSR 240 D25.

Fluid passing through heating compartment 29 becomes incident on baffles 32 and 33 before passing into the reaction compartment 30. Baffles 32 and 33 tend to break up any laminar fluid flow and generate turbulence therein to effectively mix the fluid so that it arrives in reaction compartment 30 at an homogenous temperature

Thermocouple sensor 35 provides an electrical input signal to controller 12 which corresponds to the fluid temperature in the reaction compartment 30. Temperature monitoring during operation of the thermal cycling device 10 is preferably achieved by a 30-gauge iron-constantan "J-type" thermocouple. The controller uses this information to regulate the heat coil 31 according to the predetermined temperature versus time profiles programmed therein and to actuate solenoid 18, as will be explained momentarily.

The fluid passing from the reaction compartment 30 to the return air compartment 34 must pass through sample compartment 27 (as shown in dashed lines) . Sample compartment 27 will also be explained momentarily. The fluid in return compartment 34 has been slightly cooled due to the heat transfer therefrom into samples located in sample compartment 27. The fluid in return compartment 34 is drawn through inlet opening 36 into blower compartment 28 where it is again forced, by action of the fan, out through outlet opening 37 into the heating compartment 39. Thus, the fluid chamber 11, when operating with vent door 14 closed, is a closed loop fluid chamber which continuously recirculates the fluid along a closed loop path through each compartment thereof in order to bring the contents therein to a uniform temperature. Continuous circulation of the air in the air chamber 11 allows the samples in sample compartment 27 to be brought to a predetermined temperature as quickly as possible, and then to be held at that temperature, if desired.

When the device 10 must be used to not only heat material located in the reaction compartment 27, but also to subsequently cool these materials as quickly as possible to a temperature at or above the ambient fluid (air) temperature, the controller 12 can be programmed to actuate solenoid 18 to cause vent door 14 to open and allow large quantities of ambient fluid to immediately flood the compartment 11 while heated fluid therein simultaneously escapes.

Deactivation of the heating coil 31 while continuing activation of the blower with vent door 14 open, will draw ambient fluid into return compartment 34 and from there into the blower compartment 28. The blower will then push this ambient fluid through heating compartment 29 where it will pass directly into reaction compartment 30 without being heated by coil 31. The ambient fluid then passes through the sample compartment 27 and escapes out of chamber 11 through the vent door 14. Due to the minimum thermal mass of material located in chamber 11, and the action of the blower fan, vast quantities of ambient fluid will be forced past the sample compartment 27, and from there out of the chamber 11. Thus, rapid cooling of samples or material located in the reaction compartment 27 is obtained.

The sample compartment 27 is sized so as to allow a plurality of samples, such as hollow elongate glass tubes containing a sample therein, to be easily located in a spaced apart orientation so that fluid may be evenly distributed around each sample. If desired, the sample compartment 27 may be sized and configured so as to allow insertion of a rack, basket, or the like which has been configured so as to accept a plurality of samples in uniform spaced apart configuration so as to simplify loading the samples into the sample chamber 27.

Access to sample compartment 27 is accomplished by rotation of the vent door 14 to its open position. Once the vent door 14 is rotated to approximately 90 degrees from it's closed position, the sample compartment 27 is easily accessible there through. Also, as can be seen in Figures 1-3, rotation of vent door 14 approximately 90 degrees from its closed position causes return fluid compartment 34 to be substantially closed off from the reaction compartment 30. Thus, when the device 10 of the present invention is in a "cooling" mode, ambient fluid enters directly into the return fluid compartment 34 and is forced through the blower compartment 28, heating compartment 29, reaction compartment 30, and sample compartment 27 substantially along the same path as the closed loop fluid flow path described above. The fluid is then forced out of the air chamber 11 and prevented from passing back into air return compartment 34 by the positioning of the vent door 14 between the sample compartment 27 and the return fluid compartment 34.

Thus, the vent door 14 not only allows ambient fluid to enter the chamber 11, it can also prevent the fluid from recirculating in a loop fashion through the chamber 11. Instead, fluid is forced to pass through the sample compartment 27 and then out of the chamber 11 to aid in the rapid cooling of the sample contents and chamber 11.

When the device 10 of the present invention is used for cyclic DNA amplification, repetitive cycling through different temperatures is required. Samples containing a reaction mixture for the polymerase chain reaction generally must be cycled approximately 30 times through temperature changes which correspond to the denaturation, annealing and elongation phases of the amplification process. The device 10 of the present invention, due to its novel characteristics described above, is capable of cycling samples in significantly shortened periods compared to the prior art For example, the DNA amplification application of the embodiment represented in the figures can pass through a temperature versus time profile cycle in 30-60 seconds (see Figure 5) This same cycle using prior art devices would take approximately 5-10 times longer These low cycle times have proven also to increase yield and specificity of the polymerase chain reaction over prior art cycling.

Example 1

The polymerase chain reaction was run in a 10 μP volume with 50 ng of human genomic template DNAes, 0.5 mM of each deoxynucleotide, 500 nM of each of two oligonucleotide primers GGTTGGCCAATCTACTCCCAGG (SEQ ID NO 5) and GCTCACTCAGTGTGGCAAAG (SEQ ID NO 6) in a reaction buffer consisting of 50 mM Tπs-HCl (pH 8 5 at 25°C) , 3 0 mM magnesium chloride, 20 mM KC1, and 500 μg/ml bovine serum albumin Thermus aquatics DNA polymerase (0.4 μ) was added, the samples placed in 8 cm long, thin-walled capillary tubes (manufactured by Kimble, Kimax 46485- 1) , and the ends fused with a laboratory gas burner so that an air bubble was present on both ends of each tube

The capillary tubes were then placed vertically in a holder constructed of 1 mm thick "prepunched perfboard" (manufactured by Radio Shack) The mixture was cycled 30 times through denaturation (90- 92°C) , annealing (50-55°C) , and elongation (72-75°C) to give the temperature versus time profile of Figure 5 Temperature monitoring of the capillary tubes was done with a miniature thermocouple (IT 23, Sensortek, Clifton, NJ) placed in 10 μt of deionized water and connected to a thermocouple monitor (BAT-12, Sensortek) Amplification products were fractionated by electrophoresis on a 1 5% agarose gel Specific amplification products were obtained in good yield Due to the fact that the device 10 of the present invention uses air as the thermal transfer medium instead of water, it has the advantage that heat transfer occurs through a low heat capacity medium (air) which can be warmed very rapidly

The response time for sample cooling is very fast due to the use of thin walled glass capillary tubes for holding samples, instead of plastic microfuge tubes as has been done in the past with prior art processes, and by minimizing the thermal mass of material inside the chamber 11 (see Figure 5) Such response times can allow for optimization of the time and temperature requirements for the denaturation, annealing, and elongation steps in the polymerase chain reaction

Further, shortened "ramp" times are obtained, I e , the time required to bring the temperature of the sample from one temperature level to the next temperature level corresponding to phases in the amplification process is shortened This decreases the time required for a complete amplification, as well as allowing specific study of annealing, denaturation and enzyme kinetics within a polymerase chain reaction protocol

The baffles 32 and 33 (as shown m Figure 3) may be used if desired to achieve better temperature homogeneity within the sample compartment 27 As shown in this embodiment baffles 32 and 33 decrease the temperature variation m the reaction compartment 30 from about 10°C, to about 2°C If desired, further (or more complicated) baffles may be used to further decrease the temperature variation in reaction compartment 30. Alternately, as shown in Figure 4 the fan may be positioned downstream from the heating coil 31, but before the sample compartment 27 to achieve more uniform mixing

Amplification products obtained through the use of apparatus 10 are at least qualitatively and quantitatively as desirable as those obtained through the manual water bath cycling method However, advantages in specificity and yield are possible with rapid thermal control of the reaction mixture. Figure 6 shows the effect of the temperature versus time profile of Figure 5 as used with the thermal cycling apparatus 10 on specificity (i.e.. one specific product yield as opposed to a plurality of similar or "shadow" products) . As can be seen, the shorter the ramp and annealing time, the greater the product specificity. The rapid temperature response of the apparatus 10 allows improved specificity and yield which is not possible with prior art systems.

Figure 7 shows the effect of varying the denaturation time of the temperature versus time profile of Figure 5 as used with the thermal cycling apparatus 10 of the present invention on DNA amplification yields. The brighter vertical lines each correspond to a particular time at a denaturation temperature. As can be seen, the yield is greatest at the shortest possible denaturation time. Such a result is not possible with prior art systems.

As has been shown, by decreasing the thermal capacity (thermal mass) of the apparatus 10, the present invention can markedly decrease the total time required for carrying out the polymerase chain reaction. In addition, the use of small sample volumes further shortens the total time required for the reaction and also reduces the amounts of expensive reagents which must be used by up to about 90%, thus further reducing the cost of carrying out procedures using the present invention. For example, in the embodiment represented in Figures 1-3, capillary tubes 108 having inner diameters in the range from about 0.25mm to about 1.0mm can desirably be used. In some applications, capillary tubes 108 having inner diameters in the range from about 0.02mm to about 0.1mm can also be desirably used.

The apparatus 10 of the present invention is useful for amplifying DNA from any source. Although particular configurations and arrangements of the present invention have been discussed in connection with the specific embodiments of the thermal cycling device 10 as constructed in accordance with the teachings of the present invention, other arrangements and configurations may be utilized. For example, various fluids other than air, of generally low thermal mass, may alternatively be used in the device 10. Another embodiment of the present invention is represented in Figures 8A-C. Figure 8A is a perspective view and Figure 8B is an elevational cross sectioned view of the additional embodiment. It will be understood that many of the earlier explained components and teachings also have application in the embodiment illustrated in Figures 8A-C. Thus, only the pertinent additional information concerning this embodiment will be provided below. Importantly, in the embodiment of Figures 8A-C, the heat producing element is adjacent to the biological sample containers allowing faster heating and cooling of biological samples as explained below.

As will be appreciated shortly, the apparatus of Figures 8A-C provides even greater improvement over the prior art in the speed at which thermal cycling can be carried out, e.g.. 15 or 30 cycles of DNA amplification in 30, 15, 10, or even fewer, minutes. Furthermore, the apparatus 100 provides better thermal homogenization throughout the samples than previously possible.

Shown in Figure 8A is the general configuration of the housing 102 of the embodiment. The housing 102 rests on feet 104 (best seen in Figure 8B) and functions to hold the other described structures in place and to isolate those structures which become hot from the surrounding environment. Included in the embodiment 100 of Figure 8A are input keys 25 and a display 26 as in the previously described apparatus 10. The previously described control structures can readily be modified or used as a pattern for a control means for use in the embodiment of Figures 8A-C.

As shown best in the cross sectional view of Figure 8B, a sample chamber is designated by bracket 106. A lid 138 connected to the housing 102 by a hinge 131 can be opened to allow access to the sample chamber 106. The sample chamber 106 is preferably cylindrical in shape but can be of any shape or size required by the particular application.

The sample chamber 106 is preferably lined with a black colored foam material 110 whose surface has light absorbing characteristics with the bulk of the thickness of the foam having insulating characteristics. The black foam material can be one which is readily available in the art and one fabricated from a plastic material. The foam 110 is preferably a material which is readily cooled by the air passing there over, i.e., the material has low thermal conductivity and a porous surface.

The dark or black porous surface of the material converts shorter wavelength radiation striking the surface into longer wavelength radiation, i.e., heat, which is radiated into the sample chamber.

The foam 110 functions to thermally isolate the sample chamber from the surrounding air space in the housing and also to convert the light emitted by lamp 112 into thermal energy. The foam 110 can be replaced with other structures. For example, a material having a black, dark, or other nonreflective surface, such as a thin sheet of polycarbonate having one surface painted black, can be backed by an insulative material, such as a fiberglass or foam material. The black or dark surface, which can be painted on a number of different substrates, converts shorter wavelength radiation striking it into thermal radiation while the insulative material thermally isolates the sample chamber from the surrounding environment. Thus, using the teachings provided herein, those skilled in the art can utilize many different materials and structures as a lining for the sample chamber. The lamp 112 is preferably a 500 watt halogen lamp. If appropriate control devices are used, higher power lamps or a plurality of lamps, such as four 500 watt halogen lamps, can be used. A lamp socket 112A is attached to the housing 102 by a support 112B. The lamp 112 is able to very rapidly and uniformly heat the sample chamber 106 to the desired temperature. Other sources of heat, i.e. infrared radiation, such as the earlier described nichrome wire element, can also be used within the scope of the present invention.

Represented in Figure 8B are two thin-walled capillary tubes 108 such as those described earlier. While two thin-walled capillary tubes 108 are shown, the sample chamber 106 can hold many such tubes. The thin-walled capillary tubes 108 have several important advantages over previously used devices as described earlier and, together with the sample chamber 106, function as the one presently preferred example of a means for holding a biological sample

It will be appreciated that many other structures performing equivalent or similar functions can also be used The thin-walled capillary tubes 108 are preferably left partially extending out of the sample chamber through apertures 140 for ease of access but may be completely contained within the sample chamber 106 as may numerous other fluid holding structures which are suited to particular applications The preferred thin-walled capillary tubes 108 have a capacity of about 10 μ£ As will be understood the volume of the sample should be keep small, and the surface area of the sample holding structure relatively large, and together they present a relatively small thermal mass It is also preferred that the sample holding structure contain a volume anywhere from about 1 pf to about 10,000 μf but those skilled in the art will appreciate that other volumes of samples can also be used within the scope of the present invention if the different thermal mass of the structure is considered

The lamp 112 and the insulative foam 110 together provide rapid and uniform heating of the sample contained in the thm-walled capillary tubes 108 and the air contained within the sample chamber 106 A thermocouple 134 is included within the sample chamber 106 to sense the temperature within the chamber and is used to maintain the desired temperature within the sample chamber as earlier described

The thermocouple 134 is preferably one available in the art whose thermal response substantially matches the thermal response of the biological sample and the container holding the same Such thermocouples can be commercially obtained from sources such as Idaho Labs which manufactures a line of thermocouples referred to as metal sheathed, J-type thermocouples The matching of the thermal response of the thermocouple to that of the biological sample and container can be preferably carried out by inserting a micro thermocouple, such as the model IT-23 thermocouple available from PhysiTemp as known in the art, into a typical biological sample being held by the chosen container and subjecting the sample and the thermocouple under test to the same temperature changes The thermocouple under test, or some external criteria, can be changed until the thermal response of the thermocouple suitably matches the thermal response of the sample and its container.

The arrangement represented in Figure 8B provides more uniform heating and cooling of the sample than previously available devices In previously available devices, transfer of heat throughout the sample is carried out by convection through the sample Convection induced movement of the sample within whatever structure is used to hold the sample is caused by temperature gradients or differences in the generally small biological samples (e.g . 10-100 μ()

The effect of temperature gradients within the sample become more pronounced and more difficult to control as the cycle time for a sample decreases The existence of uneven temperatures within a sample, and particularly the reliance on "mixing by convection" within the sample relied upon by the prior art devices, generally increases the cycle time for a sample and likely has deleterious effects on the biological sample The apparatus 100 is capable of providing heating and cooling such that thermal differences within a 10 μt sample are maintained at not greater than ±1°C at all times during a 30 second cycle

In order to promote uniform heating and cooling, it is preferred that the thm-walled capillary tubes 108 be at least somewhat uniformly spaced from the heat source, for example, lamp 112 in apparatus 100 Figure 8C provides a diagrammatic top view of the lamp 112 and the plurality of thin-walled capillary tubes 108 as arranged in the apparatus 100 represented in Figures 8A-B

In the arrangement represented in Figure 8C, the thin- alled capillary tubes 108 which are farthest from the lamp 112 (as indicated by line F) are preferably no more than substantially 40%, and more preferably no more than substantially 25%, farther from the lamp 112 than the distance between the lamp 112 and those thin walled capillary tubes 108 which are closest to the lamp 112 (as indicated by line N) For example, the distance indicated by line N can be about 7 3 cm while the distance indicated by line F can be about 8 5 cm It will be appreciated that the arrangement of the thin-walled capillary tubes 108 can be other than that represented in the figures, for example, circular or semi-circular. Moreover, it will appreciated that the point from which to measure the distance between the heat producing element and the sample containers will vary as the type and size of the heat producing element varies. For example, the heat producing element may comprise a plurality of lamps or electric resistive elements which vary in shape and size. In some embodiments, it may also become important to consider the distance from the sample chamber wall the sample containers are positioned. In the illustrated embodiment, the apertures 140 (see Figure 8A) function as a means for holding the sample containers but other structures performing equivalent functions can also be used in accordance with the present invention.

The apparatus 100 also cools the samples contained in the capillary tubes 108 very rapidly and uniformly In order to cool the sample chamber 106, air from outside the housing 102 is drawn into the interior of the housing through a lower housing portal 114 by a fan 116 which is connected to a motor shaft 122 driven by a motor 118. Since rapid cooling of the sample chamber is desired, it is preferred that the combination of the motor 118 and the fan 116 be able to move sufficient volumes of air into the sample chamber 106 and then disperse that air inside the sample chamber 106, as will be explained shortly Arrangements other than the motor 118 and fan 116 illustrated in Figure 8B can also be used within the scope of the present invention

The use of air as the thermal transfer medium, in contrast to other gases and liquids, has the advantages of being inexpensive, readily available, easily mixed, and never making a mess In the case of the described embodiments, the high surface area-to-volume ratio of the sample containing capillary tubes provides for rapid thermal transfer using air as the thermal transfer medium

During cooling portions of the thermal cycle, the action of the fan 116 draws ambient temperature air into the housing 102 A vent door 128, articulating on hinge 129, is provided. The vent door 128 is automatically opened by way of a solenoid 132 so that the interior of the housing 102 is sealed off from the upper housing portal 130 In some embodiments, the solenoid 132 is preferably replaced by a stepper motor as is known in the art The use of a stepper motor allows the vent door 128 to be accurately and incrementally opened and closed in accordance with the needs for heating and cooling the samples. Those skilled in the art will be able to derive an appropriate control mechanism for use with a stepper motor, for example an SC-149 stepper motor controller (available from Alpha Products) as known in the art, using the information set forth herein

Due to the arrangement of the lower sample chamber portal 120 and the larger cross sectional area and position of the upper sample chamber portal 126, room temperature air is moved into the sample chamber 106 and is dispersed and mixed within the sample chamber 106 by a paddle 124 which is connected to the motor shaft 122 The paddle 124 should rotate at a relatively high rate, for example, fast enough to create air velocities of around preferably about 250, more preferably 500, and most preferably 1000 meters per minute within the sample chamber 106 With the paddle 124, which can be a single or a multivane paddle, rotating at a high speed, air is moved, or drawn, into the sample chamber 106 and vented out of the sample chamber 106 following the path indicated by the dashed line 136 The rotation of the paddle 124 also promotes mixing of the air entering the sample chamber 106 and ensures the most efficient transfer of thermal energy from the surfaces of the thm-walled capillary tubes 108 to the air passing through the sample chamber 106 It will be appreciated that structures other than those illustrated herein can perform equivalent functions

As the solenoid 132 is actuated to open the vent door 128, all of the room temperature air moved into the sample chamber 106 is exhausted through a sample chamber upper portal 126 and then through the upper housing portal 130 carrying the heat from the sample chamber 106 to the surrounding atmosphere The rapid mixing of the air that passes through, and is disbursed in, the sample chamber 106 results in rapid and uniform cooling of the samples.

Example 2

Figure 9A shows the results of four different temperature/time profiles (A-D) and their resultant amplification products after thirty cycles (A-D) . The profiles A and B in Figure 9A were obtained using a prior art heating block device using the prior art microfuge tube. As can be seen in Figure 9A, the transitions between temperatures are slow and many nonspecific bands are present in profiles A and B. Profile B shows improvement in eliminating some of the nonspecific bands (in contrast to profile A) by limiting the time each sample remains at each temperature thus indicating that shorter times produce more desirable results.

Profiles C and D were obtained using the apparatus of Figures 8A- B. As can be seen in Figure 9A, amplification is specific and, desirably, even though yield is maximal in C (60 second elongation) it is still entirely adequate in D (10 seconds elongation) .

The optimal times and temperatures for the amplification of a 536 bp fragment of β-globin from human genomic DNA were also determined. Amplification yield and product specificity were optimal when denaturation (93°C) and annealing (55°C) were less than 1 second. No advantage was found to longer denaturation or annealing times. The yield increased with longer elongation times at (77°C) but there was little change with elongation times longer than 10-20 seconds. These unexpected results indicate that the previously available devices used for DNA amplification are not maximizing the conditions needed to optimize the physical and enzymatic requirements of the reaction.

Further information can be obtained from: Wittwer, Carl T. , Marshall, Bruce C, Reed, Gudrun B., and Cherry, Joshua L., "Rapid Cycle Allele-Specific Amplification with Cystic Fibrosis ΔF_50B Locus," 39 Clinical Chemistry 804 (1993) and Wittwer, Carl T., Reed, Gudrun H., and Rire, Kirk M., "Rapid DNA Amplification," THE POLYMERASE CHAIN REACTION 174 (1994) which are both now incorporated herein by this reference. From the information provided in Figure 9A, it can be seen that the embodiments of the present invention subject the samples placed therein to rapid thermal cycling wherein the temperature of the sample is increased and decreased at a rate preferably at least as great as 0.5°C/second In the case of the present invention carrying out the polymerase chain reaction, the temperature change is preferably carried out over an approximate range of between 30°C to 50°C It is preferred that the thermal cycles be carried out quickly enough to complete at least thirty thermal cycles in forty minutes and more preferably complete thirty thermal cycles in twenty minutes and most preferably complete thirty thermal cycles in ten minutes

The apparatus 100 more preferably increases and decreases the temperature of the sample at a rate at least as great as 1 0°C/second and even more preferably at a rate at least as great as 4 0°C/second and most preferably at a rate at least as great as 10 0°C/second Critically, the biological sample, not just the surrounding medium and/or the sample container, must undergo the specified thermal change The previously available devices, while having the drawback of not being able to perform thermal changes as rapidly as the present invention, also did not recognize the problem of changing the temperature of the sample, not just the temperature of the surrounding medium and container, rapidly and uniformly

Referring now to the chart of Figure 9B, the method of the present invention can desirably achieve thermal cycling preferably at a rate at least as great as 10°C/sec , and more preferably at a rate at least as great as 20°C/sec , over a temperature range of greater than about 20°C, more preferably over a temperature range of greater than about 30°C, and most preferably over a temperature range of about 40°C Figure 9B shows the temperature in °C of the biological sample, not just the surrounding air or container, as the biological sample undergoes thermal cycling Figure 9B shows a PCR sample beginning at about 74°C and being heated to a denaturation temperature, indicated at D, of about 92°C for 2 seconds The sample is then cooled to an annealing temperature, indicated at A, of about 55"C for two seconds The transition between the denaturation temperature and the annealing temperature covers a range of 37°C in ust under 4 seconds providing a rate at least as great as 10°C/sec. The sample is then warmed to an extension temperature of 74°C for five seconds as indicated at E in Figure 9B. The cycling of the sample through the denaturation temperature, the annealing temperature, and the extension temperature is repeated thirty times or as many times as desired

Figures 9C-G show exemplary cycles of other preferred temperature/time profiles which are achieved by the present invention It will be understood that those skilled in the art can alter the represented temperature/time profiles to carry out specific processes m accordance with the present invention Those skilled in the art will also appreciate that the previously available devices and methods, such as devices which conduct heat to and from the sample via a solid or liquid, cannot provide the resulting temperature/time profiles described herein Moreover, the previously available devices and methods do not suggest or teach the temperature/ ime profiles described herein Furthermore, it will be appreciated that the previously available devices and methods utilizing air as the transfer medium, for example previously available chromatograph c ovens, cannot provide, and do not suggest or teach, the temperature/time profiles which are described herein and obtained by the practice of the present invention

In order to provide the fastest thermal cycling time, it is preferred that the lamp (112 in Figures 8A and 8B) be rated at 2000 watts or a plurality of lamps be included which provide similar output It is also preferred to include a temperature slope control circuit which is represented m Figure 10 in conjunction with an A-bus controller/acquisition system using an 8052 micro controller board with a clock and high level program interpreter available from Alpha Products (model no SP-127) of Darian, Connecticut Exemplary programming code used in connection with the described micro controller is included in the Programming Code Appendix A attached hereto and incorporated herein The programming code provided in Appendix A is a BASIC52 file for serial downloading into the micro controller and provides exemplary temperature slope control during thermal cycling Use of the 2000 watt heat producing device and the described control structures allows thermal cycling rates of 20°C/sec. to be desirably obtained

The preferred arrangement for the temperature slope control circuit represented in Figure 10 will be explained with the understanding the additional necessary components not explicitly illustrated m Figure 10 can readily be supplied by those skilled in the art

The temperature slope control circuit of Figure 10 includes a thermocouple 200 matched to the sample temperature response as explained earlier The thermocouple 200 is connected to an integrated circuit 206, which preferably is one known in the art as an AD595, whose output is conveyed to a 4th order low pass filter 208 with a cutoff frequency of 100 Hz and to a 12 bit analog-to-digital convertor 210 whose output is used to provide a digital display of the temperature

The output of the circuit 206 is also conveyed to a measured slope circuit 212 The measured slope circuit 212 preferably includes a 353 operational amplifier 218, a 100 KΩ potentiometer 214, a l MΩ potentiometer 230, and a 22 μF capacitor The measured slope circuit 212 outputs a signal to the inverting input of a 353 operational amplifier 246

A slope set circuit 222 includes a positive slope set digital to- analog converter 226 and a negative slope set digital to-analog converter 224 The digital-to-analog converters 224 and 226 are preferably 8-bit digital-to-analog converters referred to in the art as DA147 The slope set circuit can preferably receive instructions from another digital device (not illustrated in Figure 10) such as a personal computer The output of the slope set circuit 228 is communicated to a summing circuit 240

The summing circuit 240 preferably includes 100 KΩ resistors 236, 238, and 244 and a 353 operational amplifier 242 The output of the summing circuit 240 is conveyed to the non-inverting input of the operational amplifier 246 and represents the desired slope of the temperature change. The output of the operational amplifier 246 is provided to a transistor 248 contained within a power switching circuit 262.

The power switching circuit 262 includes a 5 VDC supply 250 providing current to the transistor 248. The transistor 248 has its emitter connected to a 3010 circuit 254 by way of resistor 252 which is preferably a 330 Ω resistor. The 3010 circuit 254 includes an output connected in series with a resistor 256 which preferably is a 180 Ω resistor. A triac 258 is preferably used to control the current delivered to a lamp 262, or other heat producing device, from a source of AC current 260.

The temperature slope control circuit represented m Figure 10, in cooperation with the other described system components, provides thermal cycling of biological samples as great as 20°C/sec over a temperature range of 30°C, and most preferably over a temperature range of 40°C, with homogeneity being maintained throughout the biological sample.

It will be appreciated that the systems described herein can readily be used for many different applications including: polymerase chain reaction processes; cycle sequencing; and, other amplification protocols such as the ligase chain reaction. The present invention also advantageously provides an apparatus for accurately controlling the temperature of samples located in the sample chamber and quickly and accurately varying the temperature of samples located in a chamber according to a predetermined temperature versus time profile.

As indicated earlier, and in contrast to the teachings of the prior art, the polymerase chain reaction can be performed rapidly. Using the methods and apparatus described herein, the necessary number of temperature cycles can routinely be completed in much less time than possible with the prior art devices, for example in less than 15 minutes. By minimizing denaturation and annealing times, the specificity and yield of rapidly cycled amplifications are also improved to an extent not otherwise previously possible. Moreover, in addition to facilitating rapid heat transfer, the use of optically clear sample containers, such as clear capillary tubes, allows for continuous fluorescence monitoring of DNA amplification in accordance with the present invention.

Figure 10A shows graphically the effect of temperature transition rates on PCR reaction specificity and yield using an apparatus of the present invention. The results of Figure 10A were obtained using a 536 base pair fragment of the beta globin gene which was amplified from 50 ng of human genomic DNA with 50 mM Tris, pH 8.3 , 2 mM MgCl₂, 50 μg/mf bovine serum albumin, 0.5 μM each primer, 0.2 mM each dNTP, and 0.4 U native Taq DNA polymerase in a 10 μP reaction. The human beta-globin primers RS42 and KM29 (536 base pairs) are described in CT. Wittwer, G.C. Fillmore and D.R. Hillyard, "Automated Polymerase Chain Reaction in Capillary Tubes with Hot Air," Nucl . Acids. Res. 17:4353-4357. Temperature cycling parameters were 94°C for 0 sec., 55°C for 0 sec., and 72°C for 10 sec. Thirty five cycles of amplification were performed with the indicated rates between all temperatures. The samples were electrophoresed on 1.5% agarose gels and stained with 0.5 μg/mP ethidium bromide. Specificity and yield both decrease as the temperature transition rate decreases.

Fluorescent probes can be used to detect and monitor DNA amplification. As known to those skilled in the art, useful probes include double-stranded-DNA-specific dyes and sequence-specific probes. With the intercalater ethidium bromide, UV-excited red fluorescence increases after amplification. While microfuge tubes have been used as a sample container for DNA amplification, the embodiments of the present invention described herein advantageously utilize sample containers with many of the characteristics of structures referred to herein as capillary tubes.

The use of the sample containers described herein allows detection of fluorescence while the sample is held within the container, as will be explained more fully hereinafter. Those skilled in the art will appreciate the number of different schemes of fluorescence detection of DNA amplification which are now available. For example, sequence-specific fluorescence detection is readily possible using the present invention and oligonucleotide hybridization probes. As another example, dual-labeled fluorescein/rhodamme probes can be cleaved during polymerase extension by 5 ' -exonuclease activity, separating the fluorophores and increasing the fluorescein/rhodamme fluorescence ratio.

Using the embodiments of the present invention described hereinafter, fluorescence can be measured after temperature cycling is complete, once per cycle as a monitor of product accumulation, two or more times during a temperature transition, or continuously withm each cycle In contrast to the present invention, previously available methods only cycle relatively slowly and do not teach acquisition and analysis of fluorescence during temperature changes .

The present invention allows cycle-by-cycle monitoring for quantification of initial template copy number To carry out such cycle-by-cycle monitoring, fluorescence is acquired during the extension or combined annealing/extension phase of each cycle and related to product concentration. For example, a quantitative assay for hepatitis C RNA using the mtercalater YO-PRO l™ is known the art and can be used in accordance with the present invention For more information see Iεhiguro, T. , J. Saitch, H Yawata, H Yamagishi, S Iwasaki, and Y. Mitoma, 1995, "Homogeneous quantitative assay of hepatitis C virus RNA by polymerase chain reaction in the presence of a fluorescent mtercalater," Anal. B ochem 229 207-213 Prior to the present invention, continuous fluorescence monitoring within each cycle during temperature transitions has not been attempted

In accordance with one aspect of the present invention, one embodiment of the present invention disclosed herein is a rapid temperature cycler integrated with 2-color fluorescence optics to provide continuous fluorescence monitoring As will be more fully discussed below, different preferred fluorescence techniques for monitoring DNA amplification are provided herein as specific examples of carrying out one aspect of the present invention Those skilled in the art will be familiar with the use of ethidium bromide in fluorescence techniques which can be used in accordance with the present invention In one presently preferred embodiment described below, it is preferred that SYBR^® Green I, which is well known in the art and available from Molecular Probes of Eugene, Oregon, be used as a double-strand-specific dye.

In one presently preferred embodiment of the present invention, time, temperature, and fluorescence is acquired every 200 msec during the amplification reaction By acquiring data regularly during the reaction, the acquisition of such data reveals fine details of product denaturation, reannealing, and extension which is not available in the previously available apparatus and methods

As will be appreciated by those skilled in the art, once-per cycle monitoring of multiple samples undergoing DNA amplification is a powerful quantitative tool. Importantly, as will be appreciated by an understanding of this disclosure, continuous monitoring within a cycle can identify the nature of probe fluorescence, provide insight into DNA amplification mechanics not previously available m the art, and assess PCR product and probe melting curves to identify amplification products and mutations

Referring now to Figure 11, a schematic view of a preferred rapid temperature cycler with fluorescence detection is provided, generally designated at 300 A forced air hot air source 302 is preferably provided The forced air hot air source 302 is preferably a commercially available device including a 1600 watt heating coil and fan. A cool forced air cool air source 304 is also preferably provided The cool forced air source 304 is preferably a 2200 rp shaded pole blower available in the art from Dayton of Niles, Illinois, model no. 4C006B It is preferred that the cool air source 304 provide ambient temperature air, but it is within the scope of the present invention to utilize a means for providing fluid that is at a temperature lower than ambient air temperature

In the embodiment of Figure 11, ducts 306 and 308 connect the forced hot air source 302 and the forced cool air source 304 respectively, to a sample chamber 310 The ducts 306 and 308 are preferably corrugated black nylon tubing having a 2 5 cm diameter The duct 306 is connected to the sample chamber 310 via a port 306A and the duct 308 is connected to the sample chamber 310 via a port 308A A vent 312 and an exhaust fan 314 function to move air out of the sample chamber 310. Moreover, a means for shielding the interior of the sample chamber 310 from ambient light is integral with the sample chamber 310.

The temperature of the samples within the sample chamber 310 is preferably monitored by a tubular, metal-sheathed thermocouple 316, available from Idaho Technology of Idaho Falls, Idaho, model no. 1844, which is matched in thermal response to the samples held the preferred sample containers, for example capillary tubes Importantly, temperature homogeneity within the sample chamber 310 is achieved by mixing the air within the sample chamber 310 It is preferred that such mixing of the air within the sample camber 310 be carried out by a central sample chamber fan 318. The sample chamber fan preferably includes a 1 7 X 11 cm fan blade available from Idaho Technology, model no. 1862, and a motor available from Idaho Technology, model no 1861, which creates air velocities of at least 800 to 1000 meters per minute within the sample chamber 310. Such rapid air velocities may not be needed in all applications of the present invention but rapid air velocities promote extensive mixing and temperature homogeneity within the sample chamber 310.

Within the sample chamber 310, a plurality of samples are held in capillary tubes, some of which are indicted at 320, and are placed in a vertical orientation on a rotatable carousel 322 The carousel 322 is preferably fourteen centimeters in diameter and rotated by a 400 step per revolution stepper motor 324 controlled by a micro stepping drive module 326. The stepper motor 324 is preferably one available from New England Affiliated Technologies of Lawrence, Massachusetts, model no 2198364, and the micro stepping drive module 326 is preferably one also available from New England Affiliated Technologies, model no MDM7 micro stepping drive module, which provides 12,800 steps per rotation of the carousel 322. Still referring to Figure 11, a fluorescence excitation source 328 is provided. One preferred arrangement for the excitation path in accordance with the present invention will now be described with one preferred arrangement for the collection path in accordance with the present invention will subsequently be described. The fluorescence excitation source 328 preferably includes a 75 watt xenon arc source 328A focused with an elliptical reflector 328B. The xenon arc source 328A is preferably available from Photon Technology International of South Brunswick, New Jersey, model no. A1010, with f/2.5 elliptical reflector 328B. The power supply and other components needed to operate the fluorescence excitation source 328 are well known to those skilled in the art. Alternatively, a light emitting diode can be used as a fluorescence excitation source. Those skilled in the art will appreciate that many different excitation sources can be used within the scope of the present invention.

The radiation emitted by the fluorescence excitation source 328 is focused to about 2 mm using an adjustable iris 334 such as one available in the industry from Rolyn (Covina, California) , model no. 75.0125. The light emitted from the fluorescence excitation source 328 impinges upon a cold mirror 330, which is preferably available from

Rolyn, model no. 60.4400, and passes through heat absorbing glass 332, which is preferably one available from Rolyn, model no. 65.3130. After collimation through a planoconvex lens 336, preferably one available from Rolyn, model no. 10.0260, a 450-490 nm bandpass interference filter 338, preferably one available from Omega Optical of Brattleboro,

Vermont, model no. 470RDF40, a focusing planoconvex lens 340, preferably available from Rolyn, model no. 10.0260, and a 1 mm silica window 342, preferably available from Omega, to prevent condensation on the just described optical components during temperature cycling. Using the described excitation path, a 5-7 mm section of one capillary sample tube 320A is illuminated.

Still referring to Figure 11, the collection path for collecting the fluorescence emitted from the sample 320A will be described next. The optics of the collection path include a 1 mm silica window 344 which is placed in the optical path to prevent condensation on the other optical components. Two opposed aspheric lenses 346A&amp;B, preferably available from Rolyn, model no 17 1175, function to focus emitted fluorescence onto a 2 x 10 mm slit 348 The slit 348 can preferably be fabricated from cutting exposed X-ray film and the slit 348 functions as a spatial filter After the slit 348 (acting as a spatial filter) , the emitted fluorescence is imposed upon a 35 mm electronic shutter 350 operated via an electronic shutter control 352 The 35 mm electronic shutter 350 is preferably a Uniblitz shutter model no VS35 and the electronic shutter control 352 is preferably driver model no D122, both available from Vincent Associates of Rochester, New York A collimatmg aspheric lens 354, preferably one available from Rolyn model no 17.1175, is also provided

A filter 356 is also included when detection of SYBR^® Green I emissions is desired The filter 356 is preferably a 520-580 nm band pass filter, available from Omega as model no 550RDF60, which is preferably used for single wavelength acquisition For detection of other emissions, for example, a combination of a dichroic filter 358 and wavelength filters 358A and 358B can be used For example, for separation of fluorescein and rhodamine emissions, the dichroic filter 358 preferably consists of a 560 nm dichroic filter, preferably available from Omega, model no 560 DRLP, and a 520 550 nm band pass filter (358A) , preferably available from Omega model no 535DF30, for detection of fluorescein, and a 580 620 nm band pass filter (358B) preferably available from Omega, model no 600DF40, for detection of rhodamine. For separation of fluorescein and Cy5 emissions, the dichroic filter 358 preferably is a 590 nm dichroic filter, available from Omega, model no. 590 DRLP, and filters 358A&amp;B preferably consist of a 520-550 nm band pass filter (358A) , available from Omega, model no 535DF30, for detection of fluorescein, and a 660-680 nm band pass filter (358B) , available from Omega, model no 670DF20, for Cy5 detection. Those skilled in the art will readily appreciate that the use of other components can be readily implemented using the information set forth herein in order to accommodate other flourescent wavelengths .

Still referring to Figure 11, after being subjected to the respective filter 358A or 358B, the emitted fluorescence is focused through two planoconvex lenses 360A &amp; 360B, each preferably available from Edmund of Bamngton, New Jersey, model no 32970, and onto photomultiplier tubes 362A and 362B, respectively The photomultiplier tubes ("PMT") 362A and 362B are preferably available from Ha amatεu of Middlesex, New Jersey, model no R928, and are each enclosed in a suitable housing including appropriate circuitry, preferably one available from Photon Technology International, model no 714, with analog acquisition capabilities. A PMT and data acquisition control module 364 is also preferably provided Manual PMT shutters 366A and 366B, as known in the art, are also provided

The forgoing described optical components are preferably five centimeters in diameter and mounted in five centimeter universal lens mounts, such as those available from Rolyn, model no 90 0190 As can be carried out by those skilled in the art, many of the necessary structural components were machined from black Delrin™ using techniques known in the industry

Those skilled in the art will appreciate that the rapid temperature cycler with fluorescence detection 300 can advantageously be constructed using light emitting diodes (LEDs) and photodiodes in place of similarly functioning components represented in Figure 11 Thus, the function of the fluorescence excitation source 328 can be carried out by light emitting diodes The photomultiplier tubes 362A&amp;B can also be replaced with photodiodes Additional information regarding suitable light emitting diodes and photodiodes will be provided later herein It will be appreciated that technique sensitivity is limited by background fluorescence, most of which comes from the probes, not the detection system Significantly, stability is generally more important than absolute sensitivity

Those versed in the art will appreciate that the rapid temperature cycler with fluorescence detection 300 represented in Figure 11 includes the beneficial characteristics of a fluorimetry device with rapid temperature control, a combination nowhere suggested or taught in the art. PCR can be performed and analyzed during ten to twenty minutes of temperature cycling. The present invention's combination of 1) fluorescence monitoring within each temperature cycle and 2) analysis of the temperature and time dependence of hybridization provides advantages not otherwise obtainable.

The present invention also makes possible single-color fluorescence methods to monitor product purity and quantify template during PCR. Dyes that monitor DNA strand status are added to PCR reactions for observation during temperature cycling using embodiments of the present invention.

In order to explain some of the benefits which accrue with the present invention, specific examples using the apparatus represented in Figure 11 will now be provided. DNA amplification was performed in 50 mM Tris, pH 8.3 (25°C) , 3 mM MgCl₂, 500 g/ml bovine serum albumin, 0.5 μM of each primer, 0.2 mM of each deoxynucleoside triphosphate and 0.2 U of Taq polymerase per 5 μt sample unless otherwise stated in the following examples. Also in the following examples, human genomic DNA (denatured for 1 min by boiling) or purified amplification product was used as DNA template. Purified amplificat on product was obtained by phenol/chloroform extraction and ethanol precipitation (see D.M. Wallace 1987, Large- and small-scale phenol extractions and precipitation of nucleic acids (as described at p. 33-48, in S.L. Berger and A.R. Kimmel (Eds.) , Guide to Molecular Cloning Techniques (Methods in Enzymology, Vol. 152) Academic Press, Orlando) , followed by removal of primers by repeated washing through a Centπcon 30 micro concentrator (available from Amicon of Danvers, Massachusetts) . Template concentrations were determined by absorbence at 260 nm. A₂₆₀/A₂₈₀ ratios of templates were greater than 1.7.

In these examples, primers were synthesized by standard phosphoramidite chemistry, as known in the art, namely, using Pharmacia Biotech Gene Assembler Plus (Piscataway, New Jersey) . The 180 base pair fragment of the hepatitis B surface antigen gene was amplified using primers 5 ' -CGTGGTGGACTTCTCTCAAT-3 ' (SEQ ID NO 1) , and 5' -AGAAGATGAGGCATAGCAGC-3 ' (SEQ ID NO 2) (Genbank sequence HVHEPB) SYBR^® Green I dye was obtained from Molecular Probes (Eugene, Oregon) The β-actm primers and fluorescein/rhodamine dual probe were obtained from Perkin Elmer (Foster City, California) (no. N808-0230,) The human β-globm primers RS42/KM29 (536 base pairs) and PC03/PC04 (110 base pairs) are described in CT. Wittwer, G C Fillmore and D R Hillyard, "Automated Polymerase Cham Reaction in Capillary Tubes with Hot Air," Nucl. Acids Res. 17:4353-4357 which is now incorporated herein by reference The single labeled probes

5 ' -CAAACAGACACCATGGTGCACCTGACTCCTGAGGA fluorescein-3 '

(SEQ ID NO 3) and 5 ' -Cy5-AAGTCTGCCGTTACTGCCCTGTGGGGCAAG-phosphate-3 '

(SEQ ID NO-4) were synthesized using a fluorescein phosphoramidite (available from Glen Research of Sterling, Virginia, no 10 1963) a Cy5'" phosphoramidite (available from Pharmacia no 27-1801-02) , and a chemical phosphorylation reagent (available from Glen Research no 10- 1900) These adjacent probes hybridize internal to the PC03/PC04 β glob primer pair on the same DNA strand and are separated by one base pair Probes were purified by reverse phase C 18 high pressure liquid chromatography and homogeneity checked by polyacrylamide electrophoresis and absorbance (A₂₆₀ and the absorbance maximum of the fluorophore) . Hybridization probes (β-actin and β-globm) were used at 0.2 μM each

In the pertinent examples described herein, amplification samples of 5 μd were loaded into capillary sample tubes, some of which are represented in Figure 11 at 320 The preferred capillary sample tubes are those available from Idaho Technology, model no 1705, having dimensions of 1 02 mm O.D and 0.56 mm I D Once loaded, the capillary sample tubes were sealed with a butane flame The surface of the capillary sample tube was cleaned with optical grade methanol before it was loaded into the carousel 322 of the rapid temperature cycler with fluorescence detection 300 Control of the components represented in Figure 11 was achieved by use of a graphical programming language known as LabView (available from National Instruments, Austin, Texas) and a 12-bit multifunction input/output card 368A (available from National Instruments under the designation AT-MI0-E2) in a PC compatible computer 368 utilizing an Intel^® 80486 microprocessor running at a clock speed of 120 MHZ. Analog output channels on the input/output card 368A were used to control the sensitivity, i.e. the PMT voltage, of each of the photomultiplier tubes 362A&amp;B. Analog input channels on the input/output card 368A receive the signals from each of the photomultiplier tubes 362A&amp;B. The PC compatible computer 368, through the input/output card 368A, controls the position, rate and direction of movement of the carousel 322. For example, when multiple capillary sample tubes are loaded, the carousel 322 rapidly positions each capillary sample tube 320 sequentially at a monitoring location (the location represented by capillary sample tube 320A) for a 10 - 100 msec acquisition period. For continuous monitoring of a single capillary sample tube, the capillary sample tube is held in the monitoring position while data is preferably acquired every 200 msec, and is averaged in accordance with well-known techniques. Time, temperature, and preferably two channels of fluorescence are continuously displayed via a monitor 368B associated with the computer 368 as fluorescence vs. cycle number and fluorescence vs. temperature plots.

The carousel 322 should be positioned where maximal fluorescence and signals are acquired. When a single capillary sample tube, such as the capillary sample tube 320A, is monitored the signals are acquired every 200 msec with an integrating time constant set on the photomultiplier tube 362A or 362B, or both, at 50 msec. For multiple sample tubes, the time constant is set at 0.5 msec and the carousel is rotated once to locate the precise position where each capillary sample tube 320 provides the maximum fluorescence in each of the two channels. After positioning the capillary sample tube 320A at a location where maximum fluorescence is obtained, the sensitivity of each PMT 362A&amp;B iβ adjusted and the carousel rotated again to count and locate the position of all the capillary sample tubes 320 in the carousel 322. When only a signal fluorescence acquisition is desired once each amplification cycle during extension, each capillary sample tube 320 is sequentially positioned on the carousel 322 at the monitoring position for 100 msec. Continous acquisition for multiple tubes can also be obtained by continuously rotating the carousel 322. Temperature control programming was based upon, and modified from, a commercial rapid temperature cycler available from Idaho Technology under the trademark Rapidcycler™ using an 8051 cross compiler available from Systronics, Salt Lake City, Utah, designated BCI51 and Dallas development system (also available from Systronics under the designation DPB2) .

In practice, the temperature response of the rapid temperature cycler with fluorescence detection 300 is similar to the response obtained with the embodiment of the present invention disclosed in Figures 8A&amp;B allowing 20-30 second cycles (30 cycles in 10-15 min) as represented in the temperature vs. time chart of Figure 11A (which shows a few cycles of one preferred temperature profile) . When a double strand-speci ic fluorescent dye is present during amplification, fluorescence generally increases as more double stranded product is made. See R. Higuchi, G. Dollinger, P.S. Walsh, and R. Griffith, 1992, "Simultaneous Amplification and Detection of Specific DNA Sequences," Bio/Technology 10:413-417.

Moreover, it will also be appreciated that double strand specific dyes such as ethidium bromide or SYBR^® Green I can be used as generic indicators of amplification. SYBR^® Green I dye is preferred over ethidium bromide in many applications because it has an excitation maximum near fluorescein and often provides a stronger signal with DNA than visible excitation of ethidium bromide.

Fluorescence also depends on temperature, a confounding effect during temperature cycling that is usually eliminated by considering fluorescence once per cycle at a constant extension temperature. However, if temperature, time, and fluorescence are acquired every 200 msec during rapid cycle amplification, a three dimensional spiral is shown on the monitor 368B as represented in Figure 12. The three dimensional plot represented in Figure 12 is also projected in Figure 12A as a two dimensional plot of temperature vs. time, projected in Figure 12B as a two dimensional plot of fluorescence vs. time, and projected in Figure 12C as fluorescence vs. temperature. The temperature vs. time projection of Figure 12A repeats each cycle and provides essentially the same information as set forth in Figure 11A. Because fluorescence varies inversely with temperature, the fluorescence vs. time projection shown in Figure 12B at early cycles is a scaled mirror image of the temperature vs. time plot. As product accumulates, the fluorescence increases at all temperatures where double stranded product is present. However at denaturation temperatures, fluorescence returns to baseline since only single stranded DNA is present.

The fluorescence vs. temperature projection of double stranded dyes shown in Figure 12C eliminates the time axis and shows the temperature dependence of strand status during DNA amplification. The fluorescence vs. temperature projection shown in Figure 12C is for a 180 base pair fragment of hepatitis B virus DNA.

Another fluorescence vs. temperature projection is shown in Figure 13. The projection represented in Figure 13 is for a 536 base pair fragment of human β-globin DNA. Early cycles represented in Figure 13 appear identical, with a nonlinear increase in fluorescence at lower temperatures. As amplification proceeds, later cycles appear as rising loops between annealing and denaturation temperatures that show significant hysteresis. That is, the observed fluorescence during heating is greater than that during cooling. As the sample is heated, fluorescence is high until denaturation occurs (apparent as a sharp drop in fluorescence) . As can be seen in Figure 13, as the sample cools from denaturation to annealing temperatures, double strand signal increases rapidly. Also as can be seen in Figure 13, the fluorescence continues to increase during extension while the temperature is held constant. Double strand specific dyes can also be used in accordance with various aspects of the present invention. The strand status of PCR products can be followed with dyes that fluoresce in the presence of dsDNA. When SYBR^® Green I is present during amplification, fluorescence increases as more dsDNA is made. However, temperature cycling introduces a confounding effect because fluorescence is inversely proportional to temperature as shown in Figures 26A and 26B. As product accumulates, the fluorescence increases except at denaturation temperatures, where the fluorescence returns to baseline as shown in Figure 12C.

When multiple samples are monitored, using the rapid temperature cycler with fluorescence detection 300, once each cycle with SYBR^® Green I, a 10⁷-10⁸ range of initial template concentration can be discerned as represented in Figure 14. Figure 14A provides a legend for the indicia provided on the different plots in Figure 14, and subsequent figures, for different initial template copy number. When the data are normalized as the percent maximal fluorescence of each capillary sample tube 320, one hundred initial copies are clearly separated from ten copies. However, the difference between one and ten copies is marginal, and no difference is observed between zero and one average copies per capillary sample tube 320.

Double strand dyes depend on the specificity inherent in the amplification primers. As will be appreciated by those skilled in the art, nonspecific amplification at high cycle numbers can limit detection sensitivity to about one hundred initial template copies (see Figure 14) . With rapid cycling taught by the present invention, further improvements in amplification specificity are obtained further improving the overall DNA amplification performance.

Quantitifcation with sequence-specific probes has a similar dynamic range as double stranded DNA dyes but, as shown in the plots of Figures 15A and 15B, appear to discriminate even a single initial template copy from negative controls.

When low copy number detection and quantification are needed, additional specificity is provided by fluorescent probes that require hybridization for signal generation. Cleavage of a dual-labeled exonuclease probe is one technique which is capable of distinguishing a single template copy from a negative control as shown by Figure 15. Figure 15 show plots of fluorescence ratio vs. cycle number for different initial template copy number, according to the legend provided in Figure 14A.

Signal generation with 5 ' -exonuclease probes is dependent not only on DNA synthesis, but requires hybridization and hydrolysis between the fluorophores of the dual-labeled probe. This hydrolysis reduces quenching and the fluorescence ratio of fluorescein to rhodamine emission increases. For more information on this technique, see L.G. Lee, CR. Connell and W. Bloch, 1993, "Allelic Discrimination by Nick-translation PCR with Fluorogenic Probes," Nucl . Acids Res. 21:3761-3766 &amp; Livak, K.J. , S.J.A. Flood, J. Marmaro, W. Giusti and K. Deetz, 1995, "Oligonucleotides with Fluorescent Dyes at Opposite Ends Provide a Quenched Probe System Useful for Detecting PCR Product and Nucleic Acid Hybridization," PCR Meth. APPI . 4:357-362) .

Figure 25 shows fluorescence PCR results from a probe with five intervening bases between fluorescein and rhodamine labels. The forty- five cycle amplification was completed in 20 minutes using the rapid temperature cycler with fluorescence detection 300 of Figure 11. By monitoring the fluorescence ratio once per cycle, a IO⁹ fold range of initial template concentration could be distinguished. The amplification curves are shifted approximately 3-4 cycles for each 10- fold change in initial template concentration.

Although the final fluorescence signal is decreased when low copy numbers are amplified (presumably because of decreased amplification efficiency) , quantification between zero and one hundred copies is readily possible. The signal generated by exonuclease probes is cumulative and only indirectly related to product concentration. Hence, the fluorescence signal continues to increase even after the amount of product has reached a plateau. Using the information contained herein, those skilled in the art can formulate appropriate standards to control for efficiency of amplification and cleavage in order to carry out absolute quantification.

Fluorescence vs. temperature plots of 5 ' -exonuclease probes confirm that probe hydrolysis is the mechanism of signal generation. In Figure 16, a fluorescence vs. temperature plot of two-temperature cycling is shown with the β-actin exonuclease probe. In each cycle the fluorescence ratio varies linearly with temperature and there is little, if any, hysteresis. The signal increases each cycle during the annealing/extension phase when probe hydrolysis occurs . Although the fluorescence of both fluorescein and rhodamine decreases with increasing temperature (data not shown in the figures) , the rate of change is greater for rhodamine, resulting in an increasing ratio with increasing temperature. No temperature-dependent hybridization effects are apparent with the 5 ' -exonuclease probe.

In contrast, when the fluorescence signal is dependent only on hybridization, fluorescence ratio vs. temperature plots show a different pattern with hysteresis during two-temperature cycling, as plotted in Figure 17. The plots in Figure 17 represent the results obtained using two adjacent hybridization probes which are present, an upstream probe labeled 3 ' with fluorescein and a downstream probe labeled 5' with Cy5™. The probes are separated by a 1 base pair gap. During the annealing/extension phase of the reaction, the probes hybridize resulting in accumulating product and the CyS™ to fluorescein fluorescence ratio increasing. During heating to product denaturation temperatures, the probes dissociate between 65°C and 75°C, returning the fluorescence ratio to background levels. The change in fluorescence ratio during hybridization is largely due to an increase in CyS™ fluorescence from resonance energy transfer. The temperature dependence of hybridization can be used to detect mutations by a shift in the melting curve. Adjacent hybridization probes are also very useful for quantification, as shown in Figure 15B.

From the foregoing discussion, it will be appreciated that fluorescence monitoring during DNA amplification is an extraordinarily powerful analytical technique. Using the rapid temperature cycler with fluorescence detection 300, productive and cost efficient real time monitoring, sequence-specific detection, and quantification can be achieved in five to twenty minutes, depending on the number of initial template copies present.

Furthermore, the system and results represented in Figures 11-17 is particularly suited for continuous monitoring of a biological reaction using fluorescent dyes. For example, with precise temperature control and double-strand-specific dyes, product purity can be estimated by melting curves. With rapid temperature control provided by the present invention, absolute product concentration can be determined by reannealing kinetics. The present invention advantageously provides rapid temperature changes and strict intra- sample temperature homogeneity which is not available in the prior art. In contrast to the prior art, the preβent invention utilizes sample containers with a high surface area to volume ratio, (for example by using the preferred capillary sample tubes 320 in Figure 11) and uses air as the thermal transfer medium providing rapid control of sample temperature not otherwise obtainable. For example, sample temperature vs. time plots obtained when processing samples in the sample containers of the present invention show sharp spikes at denaturation and annealing temperatures (showing rapid temperature response) in contrast to the prior art conical plastic tubes which require several seconds for all of the sample to reach thermal equilibrium. Moreover, the sample containers of the present invention provide improved results over using etched silicon or glass chips as sample containers since the thermal cycle times and thermal homogeneity of the present invention are superior than the thermal cycle times and thermal homogeneity possible using such other structures.

Using the present invention, many aspects of DNA amplification which have heretofore been little understood are discernable . For example, product denaturation occurs in less than one second, yet the prior art calls for ten seconds to one minute of denaturation. Observing product melting by real time fluorescence monitoring with double strand dyes in accordance with the present invention (see Figures 12 and 13) shows that use of shorter denaturation times is very effective. As another example, many causes of the known "plateau effect" have been proposed, but few data are available to distinguish between alternatives. As shown in Figure 13, product reannealing is very rapid. In fact, during later cycles of amplification, a majority of product is reannealed each cycle during cooling before the primer annealing temperature is reached. This occurs with cooling rates of 5-10°C/second carried out by the present invention. Product reannealing with slower, prior art temperature cyclers will even be greater because more time is required to transition between denaturation and annealing temperature. This undesirable effect limits product yield, and is a major cause of the "plateau effect" known the art .

Furthermore, the present invention provides an inexpensive instrument that can be used in commercial applications and that continuously monitors fluorescence during rapid cycle amplification The thermal cycler of the present invention is capable of carrying out DNA amplification in no more than 10-20 minutes and the optical and detection components of the present invention discern one, two, three, or more fluorophores. The preferred embodiments of the present invention monitor a number of individual samples, for example, 24 samples (capillary sample tubes 320 in Figure 11) from once every few seconds, preferably once a second, and more preferably ten times each second.

It is within the scope of the present invention to prepare samples for processing using the known ninety-six well apparatus and the capillary sample tubes 320 which are then placed m one of the preferred embodiments of the present invention, for example, the rapid temperature cycler with fluorescence detection (300 in Figure 11) , for thermal cycling and analysis .

Advantageously, preferred embodiments of the present invention utilize fluorescence feedback for real time control and optimization of the biological process, for example DNA amplification, as the process is ongoing. Thus, with the preferred embodiments disclosed herein, the fluorescence which is detected is used to control temperature cycling. Using embodiments of the present invention disclosed herein, and using the preferred continuous monitoring techniques with dsDNA-specific dyes, extension will be terminated each thermal cycle after the detected fluorescence stops increasing. Further, in accordance with the present invention, denaturation conditions are also controlled by increasing the temperature only until the product is completely melted. Still further, in accordance with the present invention, primer annealing is monitored with resonance energy transfer between fluorescein and Cy5-labeled oligonucleotides . Moreover, using the present invention, temperature cycling of the sample is automatically terminated after a predetermined amount of product has been made.

In accordance with the present invention and as is possible using the apparatus of the present invention, rapid temperature cycling with minimal annealing and denaturation times improves quantitative PCR and increases the discrimination of allele specific amplification. Rapid cycling for cycle sequencing reduces sequencing ambiguities and minimizes "shadow banding" in dinucleotide repeat amplifications. In accordance with the present invention, for long PCR up to 35 kb, yield is improved when the sample is exposed as little as possible to high denaturation temperatures.

In contrast to the previous approach to PCR which treat PCR as three reactions, denaturation, annealing, extension, each of which occur at three different temperatures (as represented in Figure 18A) , one aspect of the present invention provides that a kinetic paradigm for PCR renders important improvements. Using a kinetic paradigm for PCR (as represented in Figure 18B) , the temperature vs. time curve consists of continuous transitions between overlapping reactions. The method and apparatus of the present invention is particularly efficient at carrying out PCR under the kinetic paradigm. Figure 18C is a graph representing different time/temperature profiles near an annealing temperature of 55°C. In Figure 18C, the solid trace shows a centrally positioned "spike" representing the temperature of response of a 10 μø sample. In contrast, the traces shown as short and long line segments in Figure 18C represent the temperature responses of samples obtained using heat block instruments. As can be seen from Figure 18C, the embodiments of the present invention produce annealing segment "spikes," with the advantages discussed herein, in contrast to the temperatures "plateaus" according to the conventional wisdom in the art.

The previously available instrumentation used for detection presented many drawbacks. Rapid, precise temperature cycling is provided by the system of the present invention described herein, m contrast to previously available instrumentation that is five to ten times slower. With the continuous fluorescence monitoring also provided by the system of the present invention, the temperature dependence of hybridization can be followed. By following hybridization during temperature cycling, the number of probes and/or spectral colors required can be minimized. That is, different products and mutations can be detected by their dynamic melting characteristics, rather than going to the trouble of synthesizing different fluorophore- labeled probes for each DNA species that is to be detected.

In order to provide an embodiment of the present invention that is most cost effective, a high intensity light emitting diode is used instead of a xenon arc source or a laser for sample illumination, and photodiodes are used for detection. Samples are loaded into glass capillary sample tubes, or alternatively into composite glass/plastic sample containers (see Figure 21A-D) in a 96-well format that does not require heat sealing. The present invention thus provides real time fluorescence analysis in a cost effective manner. Real time fluorescence control of temperature cycling improves amplification quality. For example, if the temperature of samples is increased only until denaturation occurs, product exposure to high temperatures is minimized. This increases yield by limiting product and enzyme degradation and increases specificity by limiting amplification of products with a high melting temperature. Reference will next be made to Figure 19, which provides a diagrammatic representation of another preferred embodiment of the present invention configured for continuous monitoring of a single sample. It will be understood, however, that the structures represented in Figures 19 and 20 can also be incorporated into a system which automatically processes multiple samples, such as the apparatus represented in figure 11 and as will be explained shortly herein. In the embodiment of Figure 19, a single sample holder 402 is placed in a holding bracket 404 positioned at the intersection of a temperature- controlled air stream and a linear optical path. The sample holder 402 includes a tube 402A which has many of the desirable characteristics of a capillary tube. In accordance with the present invention, different configurations of capillary tubes can be used and the tube 402A preferably has a rectangular cross section. The biological sample preferably is held at a bottom end of the tube 402A as indicated at 402B. A cap 402C is also preferably provided on the sample holder 402.

Reference will next be made to Figures 19A-19E which compare the effect of different configurations of sample containers on the temperature response of the sample itself. The temperature-time tracings shown in Figure 19E correspond to the response obtained using the sample container configurations represented in Figures 19A-C, respectively. Figure 19D represents a sample container which is less preferred for use in the present invention and is included for comparison. Using the information set forth herein, those skilled in the art can arrive at optimum sample container configurations for particular applications of the present invention. Further information regarding each of the sample container configurations represented in Figures 19A-D are set forth below.

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In the apparatus of Figure 19, an excitation radiation source 418, preferably an LED and most preferably a blue LED, is provided to illuminate the sample holder 402. The radiation emitted by the excitation radiation source 418 passes through aspheric focusing lenses 420 and an excitation bandpass filter 422 and the radiation is focused onto the sample holder 402.

The optical components illustrated in Figure 19 are preferably held in an optical housing 412. A housing 406 is also provided. A fan 408 is provided to move air through an air duct 414 and over the sample holder 402 held in the sample bracket 404. A temperature unit 410 is placed in the air flow path to provide heating or heating and cooling for the air passing over the sample holder 404. A nozzle 416 effectively directs the air over the sample holder 404.

The emissions which are given off by the sample pass through two more aspheric lenses 420 and an emission bandpass filter 424 and are received by a photo detector 426, which preferably is a photo diode. Those skilled in the art can readily provide the control components needed to advantageously operate the apparatus represented in Figure 19 using the information set forth herein.

Figures 19F and 19G are side and end views, respectively, of one preferred sample container 403 which utilizes a rectangular capillary tube 403A. The capillary tube 403A is preferably one available from Vitro Dynamics Inc. having dimensions of 1mm X 3mm X 50mm. A first cap member 403B and a second cap member 403C are held together by a screw 403D, the screw 403D also functioning as a holder for the capillary tube 403A.

Figures 19H and 191, respectively, show two possible orientations of a rectangular capillary tube 403A when detecting fluorescence of the sample contained therein. Figure 19H shows the rectangular capillary tube 403A oriented so that its edges are in line with the optical axis of the excitation and detection optics ("edge excitation and detection") . Figure 191 shows the rectangular capillary tube 403A oriented so that its faces are in line with the optical axis of the excitation and detection optics ("face excitation and detection") Surprisingly, the fluorescence signal obtained from the edge detection orientation shown in Figure 19H is about three-fold to about five-fold higher than obtained with the face detection orientation shown in Figure 191. The desirable characteristics of using the edge detection orientation shown in Figure 19H is at least partially due to total internal reflection which takes place in the capillary tube 403A which concentrates the fluorescence signal to the extremities of the capillary tube 403A.

Figure 20 shows the optical components of another preferred embodiment in accordance with another aspect of the present invention The optical components represented in Figure 20 are preferably incorporated into the thermal cycling and sample handling structures represented Figure 21, which will be more fully described shortly, but which can also be used with many different arrangements to provide monitoring (most preferably continuous monitoring) of a sample undergoing the polymerase chain reaction

In contrast to the arrangements previously disclosed herein, the optical excitation and detection paths are combined the embodiment of Figures 20 and 21, referred to herein as an epifluorescent path, rather than a linear path. In the embodiment of Figures 20 and 21, the excitation and emission radiation follow the same optical path between the capillary tube and the dichroic element used the excitation path. A capillary tube is particularly adapted for use in the embodiment of Figures 20 and 21 due to the total internal reflection (also referred to as "light piping") along the length of the capillary sample tube which is exploited to increase both excitation and emission intensities.

In the embodiment of Figures 20 and 21, to accommodate maximal light piping, the optical axis is parallel to the length of the capillary tube (paraxial) with the tip of the capillary tube positioned at the focal point. Assuming a refractive index of about 1.33 for the sample being detected, about 12.3% of emitted light is guided to the tip. It is understood that centrifuge action can be used to move the sample to the tip of the capillary tube.

Figure 22A charts the effectiveness of light piping when detecting fluorescence at the tip of the capillary tube and shows a 10- fold increase in signal intensity by viewing the tip (closed diamonds) rather than the side (open circles) of the capillary sample container. Also, as indicated in Figure 22B, the results obtained using capillary sample tubes of two different sizes and which were filled to different lengths with dsDNA stained with SYBR^® Green I are plotted. As can be surmised from Figures 22A and 22B, the observed epifluorescence increases as more sample is added to the tube, although the fluorescence efficiency decreases .

The optical properties of the emission from a capillary were investigated by stimulating fluorescence in a capillary filled with a fluorescein solution at 470 nm. The emission from a blunt end of the capillary was seen to be homogenous across the face of the capillary as opposed to concentrated in the glass as would be exepcted if the emission were the result of evanescent wave fluorescence.

The optical components represented in Figure 20 carry out paraxial epifluorescent illumination of the capillary tip, which provides advantageous results not otherwise obtainable. In Figure 20, an excitation radiation source 468 is preferably a blue LED, such as one known in the industry as a super bright LED and available from LEDtronicε. The emitted fluorescence signals are acquired by photo detectors 466A and 466B. The excitation radiation source 468 and the photo detectors 466A and 466B are supported on a mounting board 468 which also includes necessary circuitry and which integrates filters with the photo detectors 466A and 466B. A preferred mounting board is available from Ealing Electrooptics which includes 0.5 inch interference filters with high performance silicon photodiodes in T05 packages. The excitation and detection components are supported directly on the mounting board 468 with associated electronics. It is preferred that the optical components are preferably ≤l.O inches in diameter. A collimating lens 454, two dichroic filters 456A and 456B, a mirror 458, interference filters 460A-C, and aspheric focusing lenses 462A-C direct the radiation to and from the sample.

While the embodiment of the present invention represented in Figure 20 utilizes only two colors/wavelengths when performing an analysis, those skilled in the art can readily adapt the embodiment to provide three, or more, color analysis. To provide three or more color analysis, the apparatus represented in Figure 20 can accommodate additional dichroic filters and photo detectors. Moreover, it is within the scope of the present invention to allow simultaneous separation of wavelengths onto a linear photo detector array, as is available in the industry, for multicolor acquisition. When a linear photo detector array is used in accordance with the present invention, it is preferred that a prism or diffraction grating be utilized in cooperation with a lens and a photo detector array or CCD for detection of multiple wavelengths. One preferred linear photo detector array available in the industry collects 15-30 wavelength bins of 10-20 nm each between 500 and 800 nm. Various configurations of optical components, for example the Littrow autocollimating configuration for gratings used in most monochrometers, can be arrived at using the information set forth herein to arrive at the best accommodation between collection efficiency, spectral resolution and spatial requirements. The apparatus of Figure 20 will now be further described incorporated into an automated thermal cycling apparatus represented in Figure 21. Figure 21 provides a schematic representation of another presently preferred embodiment 400 of the present invention which includes rapid temperature cycling components, sample handling components, and the optical components represented Figure 20, all working together to provide fluorescence detection at the tip of the sample containers (epifluorescence) The rapid temperature cycler with epifluorescence detection 400 represented in Figure 21 provides particular advantages. It is to be understood that this described embodiment is merely exemplary of the present invention and that those skilled the art can arrive at many different arrangements for carrying out the invention claimed herein

In the embodiment represented in Figure 21, air is taken in through an aperture 470 and generally follows the flow path indicated by the lines 472. The temperature of the air, and thus the temperature of the plastic/glass sample container 450, is preferably adjusted using a 400 watt heating cartridge 474 which is preferably one available from Reheat, Inc. The heating cartridge 474 is positioned within a central duct 476 A fan 498 is provided to move the air in the indicated path 472. The fan is driven via a shaft 496 and a motor 494 The motor 494 is preferably a DC rare earth brush motor which is preferably available from Escap AG. and having a maximum rpm of 15,000 When heating the plastic/glass sample tubes 450, the heating cartridge is proportionally controlled and the fan is run at a relatively low speed (12 volts, 0 5 amp) to provide temperature homogeneity for all of the plastic/glass sample containers 450. When cooling the plastic/glass sample containers 450, the heating cartridge 474 is disabled and the motor 494 is run at a fast speed (for example with the above-mentioned preferred motor maximum speed is obtained by applying 27 volts, 1 4 amps) The fan 498 forces air into the aperture 470 and out via exhaust ports 471

In the preferred rapid temperature cycler with epifluorescence detection 400, it is preferred that twenty-four plastic/glass sample containers 450 (two of which are represented in Figure 21) be symmetrically arranged around the heating cartridge 474 and the central duct 476 The plastic/glass sample containers 450 are received by sleeves 451 which (due to their offset structure) allow for precise adjusting of the position of the individual plastic/glass sample containers 450 in a circular carousel 480. The sleeves 451 are preferably fabricated from brass. The off-axis structure of the sleeve 451 allows each sleeve 451 to be aligned so that the tip of the glass/plastic sample container 450 can be precisely adjusted to be at the optical focal point represented in Figure 21, both laterally and longitudinally, at the time that the rapid temperature cycler with epifluorescence detection 400 is fabricated.

The carousel 480 is supported on a bearing 482 above a housing 490. The carousel 480 is positioned by a stepper motor 488 provided with a drive gear 484 connected to the motor 488 via a shaft 486. The stepper motor 488 is microstepped (using a controller (not explicitly represented in Figure 21) from New England Affiliated Technologies) to provide over 10,000 steps per revolution of the carousel 480, providing precise positioning of each the plastic/glass sample containers 450. The interior of the housing 490 is provided with an insulative material 492, preferably in accordance with the previously described insulative material. Baffles 476 function to form the exhaust port 471 and to block ambient light.

Figures 21A-D provide additional detailed views of the plastic/glass sample containers 450 and will be referred to for an explanation of the preferred method of using the same. The plastic/glass sample container 450 includes a capillary tube portion 450B which is closed at one end. The capillary tube portion 450B can take many different configurations and is not limited to only a capillary tube type structure. It is, however, preferred that the volume of fluid held by the plastic/glass sample containers 450 be not more than 1 milliliter in order to promote sample temperature homogeneity and rapid thermal cycling. For example, it is preferred that the material from which the capillary tube portion 450B is fabricated have a thermal conductively in the range from about 20 to . , - cai cm about 35 in accordance with the formula ( ) X 10 . Further cm s degree C

information regarding the thermal conductivity of different glasses can be obtained from R.C Weast &amp; M.J. Astle, HANDBOOK OF CHEMISTRY AND PHYSICS, page E-6 (1982) (CRC Press) which is now incorporated herein by reference. The plastic/glass sample containers 450 are also provided with a reservoir portion 450C which is preferably fabricated from an appropriate plastic and joined to the open end of the capillary tube portion 450B. While many different materials can be used for the reservoir portion 450C, it is preferred that a plastic material be formed in a funnel-like shape and attached to the capillary tube portion 45OB.

A sample S is loaded into the composite plastic/glass sample container 450 using a pipette P, or some other appropriate instrument, either manually or using an automated process. It is preferred that the volume of the sample be in the range from about .01 μP to about 10,000 μP, more preferably in the range from about .01 μP to about 100 μP, and most preferably in the range from about .01 μi to about 10 μP, with about 5 μC being the most preferred volume. Once a sample has been added to each plastic/glass sample container 450, the plastic/glass sample containers 450 are centrifuged at low speed to place the Ba ples at the tips of the closed end of the capillary portion 450B, so that the sample forms a 0.2 - 2.0 cm column of fluid 450A as represented best in Figure 21B. A stopper 450D (which is preferably configured as a plastic plug) is then placed in the reservoir portion 450C to seal the plastic/glass sample container 450 as shown best in Figure 21C and the plastic/glass sample container 450 is placed in the sleeve 451 in the rapid temperature cycler with epifluorescence detection 400. It is also within the scope of the present invention to provide different structures to seal the capillary tube portion 450B.

The capillary tube portion 450B of the glass/plastic sample container 450 is preferably a glass capillary tube available in the industry having 0.8 mm inner diameter and a 1.0 mm outer diameter, and which is closed/sealed on one end and flared at the other end for receiving the plastic reservoir 450C. The glass/plastic sample containers 450 can be readily and economically fabricated. The shape of the tip 450E of the capillary tube portion 450B is optimized for optical efficiency. Flat tips as well as tips with various outside curvatures and inside curvature are all contemplated within the scope of the present invention. Those skilled in the art can select the most efficient configuration for the tip.

As can be discerned from Figures 21A-D, the addition of plastic loading and sealing structures to a capillary tube provides great advantages and allows efficient use of glass capillary tubes while retaining their desirable thermal characteristics. It will be appreciated that it is within the scope of the present invention to add the samples to the plastic/glass sample containers 450, and to subject the samples to centrifuging, in a 96-well format. Moreover, it is within the scope of the present invention to load the plastic/glass sample containers individually into the rapid temperature cycler with epifluorescence detection 400 and it is also within the scope of the present invention to provide an embodiment of the present invention to load the plastic/glass sample containers 450 in a 96-well format or some other format.

Advantageously, the composite plastic/glass sample containers 450 provide a convenient, inexpensive sample holder. With the embodiment of Figure 21, it is preferred that fluorescence is acquired from single samples one to ten times each second. When acquiring fluorescence from multiple samples at the preferred rate, the samples need to be moved into position by rotation of the carousel 480 relatively rapidly. With the preferred stepper motor 488 and appropriate control devices (which can be selected using the information contained herein) each of the twenty-four samples can be rapidly and accurately moved to the monitoring position represented in Figure 21.

When the flourescent signal from each sample is acquired for 100 msec, the signal variation (with repositioning) is &lt;1%. It will be appreciated that it is within the scope of the present invention to decrease the signal acquisition time, increase the transit speeds, and also observe the coefficient of variation from repeated sampling. When twenty-four samples are processed, and the carousel is rotated without stopping at a rate between one and ten revolutions per second, each sample has 0.37-3.7 msec of excitation and detection.

Using the information set forth herein, one skilled the art can select whether the flourescent signal is integrated via software or hardware. In one preferred embodiment, a graphical programming language is used in connection with the rapid temperature cycler with epifluorescence detection 400, such as one known in the industry as LabView (available from National Instruments) , which has subprograms for peak detection and integration. In another preferred embodiment, integration is done in hardware with variable integration time (user adjustable sensitivity control) so that the signals reach a level optimal for analog-to-digital conversion.

Using the rapid temperature cycler with epifluorescence detection 400 represented in Figure 21, continuous monitoring of the sample as the reaction is ongoing allows determination of temperature cycling requirements during amplification, based on continuous observation of annealing, extension, and denaturation. This is in contrast to the prior art where all cycling parameters are determined and programmed before amplification begins. In accordance with the prior art, using complementary oligonucleotides equivalent to the lowest melting primer, the annealing efficiency is controlled even during early cycles In many cases, extension and denaturation can only be monitored with dsDNA dyes during later cycles when enough product has been made Significantly, such a requirement is not usually a problem because denaturation and extension conditions are made permissive enough to amplify most products, and data from the first amplification can be used to optimize subsequent runs.

Still referring to Figure 21, a user interface and instrument control 500 can be fabricated using the information set forth herein in connection with the embodiment of Figure 11 As one preferred example of a user interface and instrument control 500, a PENTIUM™ microcomputer running the LabView programming language with a 12-bit multifunction input/output card (available from National Instruments) provides data acquisition and control It is preferred that the analog output signals be used to adjust the amplifiers associated with the photo detectors 466A and 466B. Analog input channels also measure the temperature of the samples via a thermocouple 499 as well as the flourescent detected from the sample by the photodiodes The user interface and instrument control 500 represented in Figure 21 also provides digital I/O control of the excitation radiation source 468, the direction of the stepper motor 488, the heating cartridge 474, and the fan 498.

When continuous fluorescence monitoring of PCR samples containing the dsDNA dye SYBR Green I or fluorescently labeled oligonucleotide probes can be used to monitor hybridization and melting during individual amplification cycles. This information can be used by preferred arrangements for the user interface and instrument control 500 to provide improved and customized thermal cycling conditions The benefits of using hybridization information for temperature cycling include:

(A) Ensuring that complete denaturation of the PCR product occurs with each cycle while-

Minimizing exposure to excessively high denaturation temperatures, thus, avoiding heat induced damage to the amplification products and polymerase

Increasing reaction specificity by minimizing the denaturation temperature which selects against products with a T-, higher than the intended amplification product (B) Maximizing the amplification efficiency by ensuring adequate time for product extension with each cycle while:

Minimizing the amount of time required for amplification by allowing no longer than needed to complete product extension.

Enhancing reaction specificity by selecting against products longer than the intended amplification product.

(C) Maximizing the amplification efficiency by ensuring adequate time for product extension each cycle while:

Minimizing the amount of time required for amplification by allowing no longer than needed to complete product extension.

Enhancing reaction specificity by selecting against products longer than the intended amplification product.

These would require longer than the allotted time to complete product extension.

(D) Initiating thermal cycling changes dependent on the level of fluorescence obtained or the current efficiency of amplification. For example, over-amplification and nonspecific reaction products can be minimized by terminating thermal cycling when the efficiency drops to a certain level. As another example, temperature cycling can be modified to initiate slower temperature ramps for melting curve acquisition when the fluorescence becomes detectable. This saves time because the slower ramps need not be used on earlier cycles. Other desirable changes may become evident on continued practice of the invention.

(E) Minimizing over-amplification damage to PCR product and/or initiation of melting curve acquisition before over- amplification has increased the background of nonspecific reaction products.

In accordance with the present invention, the user interface and instrument control 500 can follow preprogrammed time/temperature set points and/or, advantageously, can acquire detected fluorescence values and then use the acquired detected fluorescence values to alter or adjust one or more reaction parameters in real time to optimize the results obtained. As used herein, the term "reaction parameter" includes, but is not limited to, any parameter which is used as a basis for controlling a reaction. Such reaction parameters include, but are not limited to, denaturation temperature and time, primer annealing temperature and time, probe annealing temperature and time, enzyme extension temperature and time, and number of cycles. In general, control of the reaction is mitally based on an estimate of reaction parameters from the fluorescence data. The original fluorescence data is either acquired as a change n fluorescence over time (temperature specific rates of denaturation, annealing, and extension) , a change in fluorescence over temperature (product or probe T , or a change m extent of amplification (amplification yield and efficiency) . These rates, T_ms, and their first and second derivatives, are used to determine optimal reaction parameters such as denaturation temperature and time, primer annealing temperature and time, probe annealing temperature and time, enzyme extension temperature and time, and number of cycles .

As depicted m the high level block of Figure 22C, tasks are divided between those carried out by a portion of the user interface and instrument control 500 (which preferably can be an IBM compatible computer using programing based upon the teachings set forth herein) (Blocks 500A-500E in Figure 22C) and those carried out by the remaining components (Blocks 500A, and 500G-500S in Figure 22C) of the rapid temperature cycler with epifluorescence detection 400 It is to be understood that the block diagram of Figure 22C is merely exemplary and many different arrangements can be used to carry out the present invention.

As an example of the advantages of the arrangement shown in Figure 22C, product melting control will be discussed. A melting peak fluorescence value is acquired for the intended PCR product and a baseline fluorescence is acquired for the sample containing the reaction mixture at the temperature at which the product is seen to have completely melted. Each cycle of the reaction uses this fluorescence value as a target. The approach being described in this example uses two stages in to provide a time lag to accommodate the requirement of sending the fluorescence values to a separate PC computer. With each product melting step, the temperature is increased until the fluorescence reaches an intermediate value, then the power applied to the heating device is reduced so that a temperature ramp of approximately 3°C per second is imposed so that the PC computer has adequate time to analyze the fluorescence and convey to other components that product denaturation has occurred. The resulting time/temperature plot is shown in Figure 22D. Figure 22D shows a characteristic increase in the melting temperature after twenty cycles as the concentration of amplification product grows. This is due to the fact that product T_m is a function of product concentration.

As an example of the further advantages of the arrangement shown in Figure 22C, product annealing/extension will be discussed. During an extended hold at a combined annealing/extension temperature, the fluorescence of the sample is monitored and this information is used to ensure that adequate, but not excessive, time had been allowed for product extension. Fluorescence is monitored at ten second intervals, and if the fluoresce increased more than a preset ratio (typically 1.00 to 1.05), then the annealing/extension step is continued. Otherwise, the next product melting step is initiated. The interval of ten seconds is chosen to give a minimum of twenty seconds at the combined annealing/extension temperature.

Figure 22E shows a fluorescence/time plot which exhibits a characteristic increase in the dwell time at the combined annealing/extension temperature as the concentration of amplification product grows. This is due to the fact that as the primer concentration and polymerase become limiting more time is needed to complete product extension with each cycle.

As a yet another example of the advantages of the arrangement shown in Figure 22C, amplification plateau will be discussed. At the end of each annealing/extension step, the fluorescence value is acquired and stored. When this value increases to 1.2 times the lowest end-cycle fluorescence value and had subsequently stopped increasing below a user settable ratio (typically 1.00 - 1.02) the thermal cycle is terminated. Alternatively, a melting curve accusation step is initiated by entering a slow 0.1°C to 0.2°C/second temperature ramp through the product T_m and monitoring the fluorescence of the sample continuously. The resulting fluorescence/time plot shown in Figure 22D shows that after twenty-five cycles of amplification the ratio of cycle-by-cycle fluorescence growth fell below 1.00 and the reaction terminated. It will be appreciated that this approach can be used to acquire a high resolution melting curve for each sample. As a sample reaches its amplification plateau, a melting curve can be acquired for that sample, then regular temperature cycling can resume until another reaction reaches its amplification plateau.

Figure 22E illustrates useful temperature vs. time segments for fluorescence hybridization monitoring. Product melting curves are obtained during a slow temperature increase to denaturation. By quickly lowering the temperature after denaturation to a constant temperature, product, probe, or primer annealing can be detected. Probe melting curves are obtained by slowly heating through temperatures around the probe T_m. Those skilled in the art can readily utilize the system represented in Figure 21 to provide the necessary analysis, in real time if desired, during temperature cycling to provide heretofore unavailable information on the characteristics of the product, probe, and primer using the hardware and software described herein.

Absolute quantification of product is also advantageously carried out in accordance with the present invention. Continuous monitoring of double stranded DNA formation allows direct, absolute DNA quantification by reannealing kinetics. The sample temperature is quickly dropped from the denaturation temperature and held constant at a lower temperature that is still high enough to prevent primer annealing. The rate of product reannealing then follows second order kinetics. When different concentrations of DNA are tested, the shape of the reannealing curve is characteristic of the DNA concentration (βee Figure 26) . For any given PCR product and temperature, a second order rate constant can be measured. Once the rate constant is known, any unknown DNA concentration can be determined from experimental reannealing data. The curves can be fit by non-linear least squares regression during temperature cycling in real time using the LabView programming environment (explained previously) . Cooling is not instantaneous, and some reannealing occurs before a constant temperature is reached, but regression analysis allow for this in accordance with the present invention. The technique requires pure PCR product, but this can be verified by melting curves also obtained during temperature cycling. Quantification by reannealing kinetics is independent of signal level and not affected by sample volume differences .

Figure 28 is a schematic representation of another embodiment of the present invention which includes many of the structures included in the embodiment of Figure 21. In order to provide a succinct description of the embodiment of Figure 28, only those significant differences between those components represented in Figure 21 and those components represented in Figure 28 will be explained with the understanding that one skilled in the art can readily use the information contained herein to fabricate embodiments of the present invention. Figures 27A and 27B are cross sectional schematic views of the embodiment represented in Figure 28 in a run mode and a load mode, respectively.

The embodiment of Figure 28 is a rapid temperature cycler, generally designed at 502, with fluorescence detection at the tip of the sample containers with automatic positioning of the sample containers in two dimensions which improves the fluoresce signal which is obtained from the sample. Figure 29 is a perspective view of the exterior of the embodiment of the present invention including the components illustrated in the schematic representation of Figure 28.

As seen in both Figures 28 and 29, a removable circular sample tray 483 holds thirty-two samples. The removable circular sample tray 483 is placed into the rapid temperature cycler 502 so that it engages a carousel 481 which is driven by a motor 488. As the carousel 481 rotates, a hall effect position locator is used to precisely position the carousel 481 so that the each sample is precisely positioned over a flourimeter assembly 459. The flourimeter assembly 459 preferably includes a LED source 459A, three photodiodes 459B, focusing lenses 459C, and a filter assembly 459D. The flourimeter assembly 459 is similar in structure and function to that represented in Figure 20.

Most advantageously, the fluoπmeter is mounted on a slider bearing 493 which is moved by a lateral stepper motor 491 As the carousel 481 rotates, the composite plastic/glass sample containers 450 are precisely positioned over the fluoπmeter assembly 459 in the direction of the carousel and the position is noted by the apparatus via the hall effect position locator 495 while the lateral stepper motor 491 adjusts the position of the fluoπmeter assembly 459 is adjusted in a second dimension, and the position noted Thus, the rapid temperature cycler 502 provides for improved placement of a plurality of samples into the apparatus using a removable sample tray 483 and provides for improved detection of a fluorescence signal from a sample.

Provided in Figures 30A-30V are detailed schematic diagrams showing the preferred configuration of the electrical components of the rapid temperature cycler 502 represented in Figures 28 and 29 It is to be understood that the diagrams of Figures 30A-30V are merely one preferred arrangement for carrying out particular aspects of the present invention and these diagrams are not intended to be limiting of the scope of the present invention In order to improve the clarity of the diagrams, the notations which are commonly used the industry are maintained on these diagrams and are referenced in the corresponding parts list provided below.

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Exemplary programming code used in connection with the components of Figures 28, 29, and 30A-30V is included in the Programming Code Appendix B attached hereto and incorporated herein by reference.

In accordance with another embodiment of the present invention a handling system is provided for loading small volume sample vessels with liquid samples, particularly samples to analyzed by detection of emitted fluorescence. The sample vessel typically has a volume of less than 1ml, and it can be in the form of a tube (i.e. a capillary tube) or a "flat capillary" wherein the capillary space is defined by two spaced-apart plates or sheets sealed along their edges. The sample vessel typically has a volume to external surface area ratio of about lmm, more typically less than about 0.5. Capillary tubes having an inner diameter of less than lmm have a volume to surface area ratio of less than 0.25mm. The vessel used in accordance with the present invention is preferably formed from an optically transparent material. Preferred materials are optically transmissible for light having a wavelength ranging from about 400 to about 800 nm. The use of such material will allow the detection of a fluorescent signal generated in a liquid sample held by the vessel. Moreover the use of vessels with a low volume to surface area ratio for analyzing fluorescence from a fluorescent sample enables more efficient detection of the fluorescence due to enhanced total internal reflection.

Vessels having a high surface area to volume ratio (or conversely, a low volume to surface area ratio) can be difficult to load with liquid samples. Advantageously, the sample handling system of the present invention helps to overcome such difficulties. In accordance with one embodiment a vessel having a high surface area to volume ratio and an open end is provided with a funnel cap that fits onto the open end of the vessel to facilitate loading of liquid samples into the vessel. The funnel cap includes a first sample receiving port and a second sample transfer port and means for releasably fixing the funnel cap on the vessel so that the sample transfer port of the funnel cap and the open end of the vessel are in alignment. In one embodiment the funnel cap is of plastic or rubber construction and is formed so that the inner diameter of the sample transfer port frictionally engages the outer diameter of the vessel proximal to its open end. However, other means of coupling the funnel cap to the vessel are know to those skilled in the art and are withm the scope of the invention, including the use of adhesives, clamps, clasps and the like. In one embodiment the sample handling system further comprises a plug for frictional fit sealing engagement with the sample receiving port of the funnel cap. However any device or material that effectively seals the opening of the funnel to prevent contamination or evaporation of the loaded sample is suitable for use with the present invention.

Advantageously the vessels of the present invention can be used in a method for enhancing detection and efficiency of acquisition of fluorescence in a sample comprising a fluorophore. The method comprises the steps of placing a sample in a vessel having walls composed of an optically transparent material and defining a volume having at least first and second dimensions. The first dimension is less than the second dimension and the ratio of volume to external surface area of the vessel is less than lmm. Enhanced detection and efficiency of acquisition of fluorescence generated from the sample is achieved by detecting fluorescence along an axis substantially parallel to a wall along the second dimension of the vessel. In one embodiment, sample fluorescence is induced by fluorophore-excitatory illumination of the sample wherein the sample is illuminated along an axis substantially parallel to a wall along the second dimension of the vessel. In a preferred embodiment, optimum efficiency of fluorescence acquisition is achieved by fluorophore-excitatory illumination of the sample along the fluorescence detection axis (epifluorescent detection) , and fluorescence is detected along an axis through a wall of the vessel having the smallest surface area, preferably along an axis through the bottom of the vessel.

In one embodiment, the fluorescence of the biological sample is temperature dependent. For example the vessel may contain a sample comprising nucleic acid sequences and the fluorescent entity may comprise a double strand specific dye. As the temperature of the sample is raised to the denaturation temperature, fluorescence intensity decreases. Alternatively the fluorescent entity may comprise a pair of oligonucleotide probes that hybridize to adjacent regions of a target nucleic acid sequence, wherein one of said probes is labeled with an acceptor fluorophore and the other probe is labeled with a donor fluorophore of a fluorescence energy transfer pair. In this embodiment the vessel and the sample can be heated while monitoring the fluorescence of at least one fluorophore of the fluorescence energy transfer pair.

In accordance with one embodiment the vessel is in the form of a capillary tube or flat capillary that can be used with advantage in procedures that require thermal cycling of a sample, for example, amplification of a target nucleic acid sequence by the polymerase chain reaction. In one embodiment the capillary vessel is formed to be inserted into a sample holder of a device used for thermal cycling or a device used to detect fluorescence. The sample holder of the device may hold only a single vessel, or the sample holder may be in the form of a carousel for holding a plurality of sample vessels.

A carousel suitable for use in accordance with the present invention is shown in Figures 31A&amp;B. The carousel 1 is generally in the form of a disc 2 having a top surface 3, a bottom surface 4 and an outer edge 5 extending therebetween. The disc 2 has a plurality of sets of radially aligned sample receiving ports 6A, 6B, and 6C in the top surface 3, a sample vessel port 7 in outer edge 5 and a sample passageway 8 communicating with the sample receiving ports 6A, 6B, and 6C and the respective sample vessel port 7. The carousel 1 is shown with fixed sample vessels, some of which are indicated at 9 The sample vessel port 7 and sample passageway 8 are formed for receiving and fixing sample vessel 9 to the disc 2. In one embodiment the sample vessel 9 is releasably fixed to the carousel 1 to allow the removal of the sample vessel and its replacement with another sample vessel to allow for multiple use of the carousel 1. In an alternative embodiment the sample vessels 9 are permanently fixed to, or formed as an integral component of, the disc 2. In one embodiment the sample vessel 9 is fixed to the disc 2 by frictional contact between the sample vessel 9 and at least a portion of the sample passageway 8 proximal to said sample vessel port 7. Other conventional means for fixing the sample vessel in communication with the sample vessel can be used. For example, complementary screw threads can be formed on the surface of the sample passageway 8 and on the exterior surface of the sample vessel 9. In addition adhesives or any other fixation means known to those skilled in the art can be used in accordance with the present invention to fix the sample vessel 9 to the disc 2. The top and bottom surfaces of the carousel of the present invention are preferably formed to allow multiple carousels to be stacked one on top of another so that a stack of multiple carousels can be releasably engaged with a motor drive shaft and rotated simultaneously as a unit as shown in Figure 32. The embodiment shown in Figure 32 includes a stepper motor 504 and a drive shaft 506 which functions to hold and rotate the carousels generally indicated at 1. A chamber fan 508 is used to generate the air flow indicated by the arrows 512. A heating device 510 functions to heat the air which passes by the sample vessels 9. A fluoπmeter assembly 514 includes an LED source 514A, photodiodes 514B, focusing lenses 514C, and a filter assembly 514D. A fluoπmeter stepper motor 516 functions to move the fluorimeter assembly 514 in the direction of arrow 518. Those skilled in the art can readily fabricate embodiments of the present invention fashioned after the arrangement represented in Figure 32 using the information set forth herein.

In another embodiment (not shown) the carousel comprises a disc having a top surface, a bottom surface, an outer edge extending therebetween, a sample receiving port in the top surface, a sample vessel port in the bottom surface and a sample passageway communicating with said sample receiving port and the sample vessel port . The sample vessel port and sample passageway are formed for receiving and fixing a sample vessel to the disc. Preferably the sample vessels are held at a radially extending acute angle to the bottom surface of the disc. In one embodiment the sample passageway of the disc comprises a first portion having a central axis substantively parallel to the top and bottom surfaces of the disc and a second portion having a central axis forming an acute angle with the top and bottom surfaces of the disc. In this embodiment the sample vessel port and sample passageway are formed for receiving and fixing a sample vessel to the disc such that the sample vessel extends from the disc at an acute angle relative to the bottom surface of the disc .

Carousel 1 is further provided with means for closing the sample receiving ports 6A, 6B, and 6C The closure means can be a plug (not shown) that fits into the sample receiving port 6 and frictionally engages the adjacent walls of the sample passageway, or for example, adhesive backed tape, for application to the top surface to effectively seal the opening of the sample receiving port to prevent contamination or evaporation of a loaded sample. Carousel 1 is releasably engaged with a drive shaft for rotation. Any suitable engagement means well known to those of ordinary skill in the art can be used including frictional engagement, or the use of screws, bolts, locking pins or clamps. In one embodiment, the disc 2 is formed as ring having a center hole formed for receiving a drive shaft (see 506 in Figure 32) . The end of the drive shaft is preferably provided with structures for holding the discs 2 in place. The carousel 1 of the present invention can be used to deliver a liquid sample to a sample vessel 9. In one embodiment the sample vessel 9 is a capillary vessel containing a predetermined mixture (for example a reagent mixture) that interacts with one or more components of the introduced sample. In accordance with one embodiment the predetermined mixture is added to the sample vessel before positioning a capillary sample vessel into the sample vessel port. Alternatively the sample vessel is prepackaged with a predetermined mixture. The predetermined mixture may comprise reagents that react or interact with the sample to produce a detectable signal or to produce a derivative product.

The sample passageway 8 of the carousel 1 are optionally provided with one or more barriers 10 that prevent a liquid sample delivered through sample receiving ports 6A, 6B, and 6C from flowing to the sample vessel port 7 absent a biasing force on said liquid sample. The term "barrier" is used herein to include any structure that impedes the free flow of a liquid sample delivered into a sample receiving port to the sample vessel port. Examples of suitable barriers for use in the sample passageway of the carousel of the present invention include depressions or wells formed in the sample passageway, sample passageway narrowing projections or annular rims that extend from the surface of the sample passageway, porous membranes, directional valves, or flaps that are biased in a closed position.

The barriers are formed so that the liquid sample can overcome the barrier by application of a biasing force on a liquid sample present in the sample passageway and blocked by the barrier. The application of biasing force on the sample is preferably provided by the centripetal force generated by rotation of the carousel. Therefore, in a carousel having a plurality of sets of sample receiving ports 6A, 6B, and 6C in the top surface, each set with a corresponding sample passageway and sample vessel port, samples can be added individually to the various sample receiving ports and the barrier will localize the liquid sample and prevent the samples from flowing to the respective sample vessel ports. After all of the samples are delivered into the respective receiving ports, the carousel is rotated to deliver the samples to the respective sample vessel port and into an attached sample vessel.

In accordance with one embodiment, each sample passageway of the carousel communicates with a single sample vessel port and a plurality of sample receiving ports. In accordance with that embodiment, the sample passageway can optionally include a central passageway that branches to communicate with multiple sample receiving ports, or alternatively, as illustrated in Figures 31A&amp;B multiple sample receiving ports 6A, 6B, and 6C are aligned along a common axis that extends radially from the center of the disc, each of said ports communicating through one passageway with a sample vessel received in the sample vessel port. The sample passageway can be provided with one or more barriers 9A that prevent a sample added to any one of the plurality of sample receiving ports from flowing to the sample vessel port absent a biasing force on said liquid sample. Furthermore, each sample passageway can be provided with multiple barriers, each of which require a different amount of biasing force to transfer a sample over the barrier. In accordance with this embodiment, after delivery of the samples to the respective sample receiving ports, individual samples can be selectively transferred to the sample vessel port and into the sample vessel by controlling the rate of rotation of the carousel.

For example, a first sample can be delivered into a first sample receiving port and a second sample can be delivered to a second sample receiving port wherein the first and second sample receiving ports communicate with a common passageway and the first and second sample receiving ports are each provided with a barrier that prevents flow of the respective first and second sample. The barriers allow the disc to be provided as part of a kit with predetermined amounts of selected reagents, catalysts, enzymes, oils, etc. being preloaded into the sample passageway via one or more of the sample receiving ports .

In one embodiment the barrier for the second sample receiving port is formed so that a greater biasing force must be applied to the sample delivered to the second sample receiving port to pass its associated barrier than is required for a sample delivered to the first sample receiving port to pass its associated barrier. In accordance with this embodiment, rotation of the carousel at a first rate will deliver the first sample to the sample vessel port and into the sample vessel, while the second sample is prevented from flowing to the sample vessel port and into the sample vessel. Rotation at a increased second rate will then enhance the centripetal force on the second sample and result in the delivery of the second sample to the sample vessel port and into the sample vessel. Based on this principle, different samples can be delivered to multiple sample vessel ports that communicate with a common passageway and after all the samples have been loaded, the individual samples can be delivered to the sample vessel port and into the sample vessel one at a time or simultaneously by controlling the rate of rotation of the carousel. In one embodiment a first sample, comprising a fluorophore is added to a first sample vessel port and a second sample comprising oil is delivered to the second vessel port. The carousel is rotated to deliver the first sample into the sample vessel followed by the oil. The oil (or another liquid that effectively seals the first sample within the sample vessel) functions both to decrease evaporation of the first sample and to reduce the risk of contamination of the first sample.

In one example a multiple sample carousel is used to handle multiple samples simultaneously. The carousel is a disc-like structure having a multiplicity of sample receiving ports in the top surface of the disc structure and in fluid communication with corresponding sample vessels attached to the disc. Samples added to the sample receiving ports are transferred to their corresponding sample vessels by rotation of the carousel. The carousel can also have multiple sample receiving ports communicating with each individual sample vessel. Reagents can be placed by the user into a second sample receiving port that communicates with the sample vessel for delivery to the vessel with another sample that was added to the first sample receiving port, or alternatively, predetermined reagents may be located in a second sample receiving port by the manufacturer; i.e. where the carousel, the sample vessels and the predetermined reagent are in a prepackaged form. The reagents, with the sample, are delivered to the sample vessel by rotation of the carousel. An oil for overlay of an aqueous sample may be placed in a third sample receiving port that is in liquid communication with the sample vessel (and the first and second sample receiving ports) , or the oil may be added to the carousel by the manuf cturer.

Alternatively, a sample, reagents and oil for sample overlays can be delivered to a single sample receiving port. The carousel can be rotated to deliver each composition or sample to the respective vessel before a second or subsequent sample or other composition is delivered to the sample receiving port.

One preferred sample vessel carousel of this invention includes three sample receiving ports preferably, but optionally, arranged in radial alignment and in fluid communication with a common sample vessel. In accordance with this embodiment, about 1 to about 5 μP of an oil overlay, preferably dyed black, is present in prepackaged form, or delivered to the radially innermost sample receiving port. The oil overlay comprises mineral oil and about 0.01% to about 1% organic black dye such as Waxoline^® Black OBP available from Zenica, Inc. of Wilmington, DE. About 1 to abut 9 μP of a reagent master mix is present in prepackaged form or is delivered to the radially outer most sample receiving port. The reagent master mix comprises a portion of, or all the necessary reaction components. A liquid sample containing the template nucleic acid to be tested is delivered manually or robotically into the radially intermediate sample receiving port . The disc is then rotated at a rate that transfers the sample to the reagent compartment, but at a rotated rate insufficient to deliver the mixture into the sample vessel. The sample and reagent can optionally be mixed by rapid changes in the rate of the rotation of the disc. The disc is then rotated at a higher rate that causes the sample and reagent mixture, but not the oil, to move into the sample vessel. The disc is then rotated at still a higher rotation rate to deliver the oil overlay to the sample vessel. The oil will overlay the aqueous sample because of its lower density and will block light passage because of its dye content. The selective transfer of oil, reagents and sample by altering the rate of carousel rotation is achieved by a combination of: 1) varying the diameter of the fluid communication passageways; 2) varying the size or shape of the physical barriers present in the fluid communication passageways; and 3) by using the dependence of centrifugal force on the varying distance (radius) of each sample receiving port from the center of the disc.

The carousel of the present invention can be releasably engaged with the drive shaft and a motor (506 and 504, respectively in Figure 32) for rotating the carousel. Furthermore, individual carousels of this invention can be stacked upon one another and engaged with a drive shaft for simultaneous rotation (as shown in Figure 32) . In accordance with another aspect of the present invention a device is provided for monitoring the fluorescence of a sample held within a sample vessel (see 514 in Figure 32) . The sample vessel comprises an optically transparent material and has walls defining a volume having at least first and second dimensions wherein the first dimension is less than the second dimension and wherein the ratio of volume to external surface area of the vessel is less than lmm. In one embodiment the device comprises a chamber, a sample vessel holder, a light emitting source mounted in said chamber and positioned to illuminate the sample vessel along an axis substantially parallel to a wall along the second dimension of the vessel and a light detector mounted in said chamber and positioned to measure fluorescence from the sample vessel along an axis substantially parallel to a wall along the second dimension of the vessel. The light emitting source and the light detector accordance with one embodiment are mounted on a platform that can be raised and lowered (as indicated by arrow 518 in Figure 32) . In this embodiment, the light emitting source and the light detector can be positioned to measure fluorescence from the sample vessels (along an axis substantially parallel to a wall along the second dimension of the vessel) of multiple carousels when individual carousels are stacked upon one another and engaged with a drive shaft for simultaneous rotation (see Figure 32) .

In one embodiment the sample vessel holder comprises a carousel for holding a plurality of capillary tubes, and the carousel is rotatably mounted in said chamber. The light emitting source is positioned to illuminate the capillary tube through the bottom of the tube and the light detector is mounted to detect fluorescence through the bottom of the capillary tube. In addition the device is provided with a stepper motor for rotating said carousel and means for coupling the carousel to the motor.

In accordance with one preferred embodiment, the chamber of the fluorescence detecting device is further provided with a heater (see 510 in Figure 32) and a fan (see 508 in Figure 32) mounted in said device and in air flow communication with the chamber, and a controller therefor, for rapidly cycling the temperature of the chamber using, at least initially, predetermined time and temperature parameters. The device is capable of conducting polymerase chain reactions in the sample vessels held by the carousel. In particular the device allows for an improved method of conducting PCR reactions because the progress of the reaction can be monitored in real time, and thus allow the adjustment of temperature and time parameters during the course of the reaction to optimize the yield and purity of the amplified target nucleic acid sequence.

Further, in accordance with the present invention, there is provided an improved method of amplifying a targeted nucleic acid sequence of a biological sample comprising the steps of adding to the biological sample an effective amount of two nucleic acid probes that hybridize to adjacent regions of the target sequence, one of said probes being labeled with an acceptor fluorophore and the other probe labeled with a donor fluorophore of a fluorescence energy transfer pair such that upon hybridization of the two probes with the target sequence, the donor and acceptor fluorophores are within 0 to 15 nucleotides, and more preferably within 1-5 nucleotides of one another, amplifying the targeted nucleic acid sequence using polymerase chain reaction, illuminating the biological sample with a selected wavelength of light that is absorbed by said donor fluorophore during the polymerase chain reaction monitoring fluorescent emissions from said sample, and adjusting the temperature and time parameters in accordance with the data generated from the monitoring step.

Thus in accordance with the present invention an improved device is provided for conducting PCR reactions. The device comprises a chamber, a heater and a fan mounted in said device and in air flow communication with the chamber, carousel for holding a plurality of sample vessels. The sample vessels used in conjunction with this device comprise an optically transparent material and walls defining a volume having at least first and second dimensions wherein the first dimension is less than the second dimension and wherein the ratio of volume to external surface area of the vessel is less than lmm. The carousel is rotatably mounted in the chamber. The device further comprises a light emitting source mounted in said chamber and positioned to illuminate at least one of the sample vessels along an axis substantially parallel to a wall along the second dimension of the vessel and a light detector mounted in said chamber and positioned to measure fluorescence from at least one of the sample vessels along an axis substantially parallel to a wall along the second dimension of the vessel. Furthermore, the device can be equipped with a stepper motor for rotating the carousel to position the respective capillary tubes held by said carousel for illumination and fluorescence detection. Monitoring the PCR reaction in real time and determining at least one reaction parameter in accordance with the detected fluorescence allows for the adjustment of the reaction conditions to optimize the reaction. In a preferred embodiment one or more values representative of the status of the reaction are displayed in a visually perceptible manner in real time.

The carousel of the present invention can also be used for delivering a liquid sample to a capillary sample vessel. The carousel comprises a disc having a top surface, a bottom surface and an outer edge extending therebetween, a sample receiving port in the top surface, a sample vessel port in the outer edge and a sample passageway communicating with the sample receiving port and the sample vessel port. The sample vessel port and the sample passageway are formed for receiving and fixing a sample vessel to the disc. The method of using the carousel to deliver a liquid sample to a capillary sample vessel comprises the steps of selecting a carousel for receiving a liquid sample and holding a sample vessel, delivering the liquid sample into the sample receiving port of the carousel, positioning a capillary sample vessel into the sample vessel port, and rotating the carousel to deliver the sample into the capillary sample vessel.

The present invention is also directed to a system for detecting the presence of a target nucleic acid sequence in a sample. The system comprises a pair of oligonucleotide probes that hybridize to adjacent regions of the target nucleic acid sequence, wherein one of said probes is labeled with an acceptor fluorophore and the other probe labeled with a donor fluorophore of a fluorescence energy transfer pair. Preferably, the donor fluorophore emission and the acceptor fluorophore absorption overlap less than 25%, the acceptor fluorophore has a peak extinction coefficient greater than 100,000 M 'cm ¹ and upon hybridization of the two probes with the target sequence, the donor and acceptor fluorophores are within 15 nucleotides of one another. In another embodiment the donor fluorophore emission and the acceptor fluorophore absorption overlap less than 20% and upon hybridization of the two probes with the target sequence, the donor and acceptor fluorophores are within 5 nucleotides of one another, and more preferably within 3 nucleotides of one another.

In view of the foregoing, it will be appreciated that the present invention provides an apparatus for accurately submitting biological samples to thermal cycling and for quickly and accurately varying the temperature of biological samples, most advantageously adjusting one or more reaction parameters in real time or according to a predetermined temperature versus time profile. The present invention also provides an apparatus suitable for subjecting a number of different biological samples to rapid thermal cycling and also provides a thermal cycling apparatus having a thermal transfer medium of low thermal mass which can effectively subject samples to a large temperature gradient over a very short period of time.

Moreover, the present invention provides an apparatus which can subject a biological sample to rapid thermal cycling using air as a thermal transfer medium and which provides a system and method for performing PCR rapidly and for simultaneously monitoring the reaction. Still further, the present invention also provides a system and method for performing PCR rapidly and also continuously monitoring the reaction while it is ongoing and for adjusting the reaction parameters while the reaction is ongoing.

Information regarding an On-line DNA Analysis System with Rapid Thermal Cycling is found in U.S. Patent Application serial no. 08/381,703 filed January 31, 1995 which is now incorporated herein in its entirety.

The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.

PROGRAMMING CODE APPENDIX A FOLLOWS 320 WS=(DS*10+W9*9)/10tTX=»INT(WSf0.5) :GOSUB 15

325 XBY(65508)=TX:GOSUB 50.IF* TP&gt;TX3 GOTO 340

330 IF TXODS*1000T0 320

333 GOSUB 30

335 OOSUB 50: IF TP&lt;TG XJΪO 335

340 TG=DTEMP.ED= IME

345 WS=(6*WSrlO*(TG-TP))/7 :TX=-INT(WS+0.5)

350 IF TX&gt;=0 THEN GOSUB 25:XBY(65508)»TJ .XBY(65509)β4

355 IF 'I3&lt;0 TIEN GOSUB 20tXBY(65508)=0:XBY(65509)=4-TX

360 IF V=l THEN IP DTIM*5&gt;(TIME-EB) GOTO 380

370 IF DTIM&lt;(TIME-EB) GOTO 500

3ϋϋ GOSUB 50 : GOTO 345

500 PRINΓ ^•Λ\:XBY(G5500)»0ιTα=ΛTEMP

04!5 $(0)-"opl 200/gol nd;hfl 2;w£o ».x.. fβl'iGOSUB 3: ran open

855 XB (65509) ΛS*10

860 GOSUB 30

890 EB=TTMS:WS=-AS*10

913 WS-(4*WS+20*(Ta-TP))/5ιTX-XN ( S+0.5)

915 IF TX&gt;=0 'IHEN GOSUB 25 :X0Y( 65508) =TX«XBY(65509) =4

918 IF TX 0 TliEN GOSUB 20:XBY(65508) »0:XBY(65509) = -TΛ

920 IF ΛT1MI1&lt;(TΓHE-EB) aOTO 925

924 ⁽X)SUB 50 : GOTO 913

925

930 REM CLOSE

935 5 :XHY( 65508 ) «TX

937

940 <img file="WO9746712A2_D0011.tif" />

950 GOSUB 50 i IF TP&lt;TO-1 GOTO 950

<img file="WO9746712A2_D0012.tif" />

1055 χnγ(65508)--0:XBY(65509)*=255

1060 INPUT 'STRAND RΣLΛIWEAIilrø (Y/N) 7",$(1)

1070 IF ΛSC($(l).l)-78 GOTO 1290 lioo PRΓN -STRAND REΛNNEΛLΓNG IN PROCESS...'

<img file="WO9746712A2_D0013.tif" />

1140 IF TXoDS»10 GOTO 1120

1150 GOSUB 30

1160 GOSUB 50:IF TP&lt;T3 GOTO 1160

1170 EBaTIME

1180 S=(11*WS+10*(TG-TP))/12 ιTX=INT(WS+0.5)

1185 IF TX =0 THEN GOSUB 25:XBY(65508)«=TX-XBY(65509)«4

1190 IF TX&lt;0 THEN GOSUB 20:XBY(65508)«0:XBY(65509)"4-TX

119J IF 5&lt;(TTME-EB) GOTO 1200

1196 OOSUB 50 : GOTO 1180

1200 XBY(65508)=0ιXBY(65509)"255iWS=0

1210 DO: GOSUB 50:UNTIIi TP&lt;75

<img file="WO9746712A2_D0014.tif" />

1260 IF 60&lt;(T1ME-En) GOTO 1200

1270 GOSUB 50 : GOTO 1230 NEW

1 GOTO 60:PJEM ***I/0 stepper (I=$(0), 0=z(72))***

3 IF (XBY(65507).ΛND.64)=0 THEN Z(0)»XBY(65506) j GOTO 3

4 DO : WHILE (XBY{65507) .AND.128)=128

5 1-0 : DO : 1=1+1 • XBY(65506)=ΛSC($(0) ,I)

6 WHILE ΛSC($(0) ,I)&lt;&gt;13 : RETURN

7 DO i WHILE (XBY(65507).ΛND.64)=64

8 1=0 : DO : Z(I)= U3Y(65506)

9 1=1+1 : UNTIL 2(1-1)=32 I RETURN

10 REM ***error check***

15 IF TX&lt;0 THEN TX-0

16 RETURN

20 IF TX&lt;-251 THEN TX—251

21 RETURN

25 IF TX&gt;251 THEN TXc251

26 RETURN

30 GOSUB 50ιTPl=TP:Tl=TTJME

32 DO:UNTIL TIME&gt;Tl+0.2

34 GOSUB 50:TP2=TP:T2=TIME

36 SL^«(TP3-TP1)/(T2-T1) jIF Θ &lt;&gt;0 THEN ITa(τα-TP2)/SL

30 PπiNT USIN^Q(»##.##),"SL-\SL, "IT=",IT, "AT TEMl?a^»,TP2

40 IF IT&gt;1 GOTO 30

42 RETURN

49 REM **"temperature measurement***

50 E=XBY{65504) :F= CBY(65505).α*XBY(65504):TP&lt;=(F*256+G+300)/3β.3: RETURN (72)

<img file="WO9746712A2_D0015.tif" />

80 PRINT USING(###),^■#", AB (20) , ^•TEMP-.TAB (30) , "TIME".TAB (40) , 'SLOPE*

82 PRINT "1) DENATURATION', TAB(20) ,DTEMP,TAB(30) ,DTIM,TAB(40) ,DS

84 PRINT "2) ANNEALING", AB( 0),ΛTEMP,TAB(30) ,ΛTIM,TΛB(40) ,AS

B6 PRINT' "3) EXTENSION',TAB(20) ,ETEMP,TΛB(30) ,ETIM,TΛB(40) ,ES

88 PRINT *4) CYCLES ",CYCLE: RINT

110 INPUT "TYPE # TO CHANGE OR 0 IF OK (CHOICES ARE 0,1.2,3, OR 4)?^«,J

114 IF J-.0 THEN GOTO 170

116 ON J GOSUB ,120,130,150,160

118 PRIN iGOTO B0

130 IMPUT 'ENTER DENATURATION Terro_r Time, Slope: ",DTEMP,DTIM,DS

125 IF DTEMP 98 GOTO 120

126 nrrπjnN

130 NPUT "E JTER ANNEALING Tβcrj?, Time, Slopβi ',ΛTEMP,ΛTIM,AS 40 IP AS&lt;0 GOTO 130 42 IF ΛS&gt;21 GOTO 130 45 RETURN 50 INPUT 'ENTER EXTENSION Terrp, Time, Slope: \ETEMP,ETIM,Eg:RETURN 60 INPUT "ENTER Number of Cycles: * ,CYCLE:RETUR 70 $(0)*.'m3rl 500,βpl 200;hfl 2;rt2 l;esr" : GOSUB 3 : GOSUB 7 75 FOR 1=0 TO 10 : PRINT CHR(Z(I)), : NEXT I : PRINT 00 $(0)="gol ndjhfl 2;wfo .x.. }si":GOSUB 3ι rem open 10 PRINT "WAIT FOR COOLING" |XBY(65504)=3 20 DO : GOSUB 50 : UNTIL TP&lt;45 30 PRINT "TEMP=.\TP,* LOAD SAMPLES AND THEN PRESS A KEY" 40 W=GET i IF =0 GOTO 240 00 05 CLOSE 10 15 <img file="WO9746712A2_D0016.tif" /> 1280 XBY(65508)=0JXBY(65509)=255

1290 INPUT ^•ANOTHER RUN (Y/N) ?",$(1)

1300 IF ΛSC($(1),1)-7B GOTO 2000

1400 PRINT:$(0)=-"gol ndjhfl 9;w£o =,x..&gt;βl"iGOSUB 3:GOTO 80

2000 $(0)-»'øoi ndjhfl 9; fo -.X..rsl^#:GOSUB 3:END

PROGRAMMING CODE APPENDIX B FOLLOWS

10

Temperature processor(IC 2) hex file (Temp24a.hex):

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200O3300C2AFC2D4C2D3752100752200752300D215D28AAF81758130858115CODOCOFOC098 20005300E0C08753C6FB1230897527405388CF5389F012317D78FC12305D12324912365439 20007300C20EC20FC208759850D2ACD2AFE587DOEODOOOD001DOD030E20530E4028012 206A

20009300E602800DBF070280089038A974FA02373975C7AA75C755438740907471C082C03F 2⁽X⁾OB3008378187AOOCOO⁽⁾Cω29037A6C082CO83DOO3DOOlDOO5DOO4D002D0OO123O959OD6 2000D30O7084C082C08378O07A0ODO83DO82E8F0907060C082C08378007AO0D083D082E8C 1 2000F300F0A3EAF002013BD281D29090705CC2D57400C4C0A8C2AFC282C2907908C282 A28C

:2001130O81339280D282D9F5F5FO79O4C282A28133D282D9F7C282D290D0A8F0E5F0C454C9 :200133000FA3F030D501222202015BD2A3C2A122D2A3D2A122C2A3D2A122C2A222D2A 22248

20015300D2A022C2AO22222212015612014812015012015A907055C082CO8378017A00D0FF 2001730083D082E8F078007A001230D278007A0012319D 123216A8887A00C000C00278F7F2 200193007 A0OO00()C002.XX)3IX)01DO02I^

2001B30000C002IX)03D001D(X)2D000123231888890232FA882AA83123235D217907471127D 2001D3O033931233D8123416D297907O87CO82CO8378007AO0D083DO82E8F0122328122357 2001F3002890705AC082C08390705CEOF8A3EOFAD083D082E8FOA3EAF01225FF907087 E09D

2( i3WF87AOOC(X)OC00278007AOOCOOOC002D003D001D002D00012342FE84A6003020110 20023300F1122328120153120143907O7EC082CO8378O07A00DO83D082E8FOA3EAF09O7OB2 200253007CC082C08378007AOOD083D082E8FOA3EAF0907088C082C08378017AOOD083D07B 2002730082E8F078007A001230D2907074C082C08378017AOOD083D082E8FOA3EAF0907025 2002930062C082C0839O7060E0F8A3E0FAD083D082E8F0A3EAF0907060C082C0839O708947 2O02B3OOCO82C0839O7O74EOF8A3EOFAC0O0C00278087AO0COO0CO02DO03D0O1DOO2DOOO99 20O2D3O012348EC()O0CO02C(X)0C00278E87A03COO0C002IXX)3D()OlD0O2D0OO1235O8E84AOD 2TO2F3()07W30230C278017AOOC()OOC002D003DOM^

2O0313O0DO02DOO012350ECO00COO2DO03DO0189828B83123528DO83D082E8FOA3EAFO9O9B 20O3330O7O8OC082C083907089C082CO83907O74E0F8A3E0FACO00CO0278087A00C0OOCO62 2003530002D003D()01DW2D0001234CFC(X)()C00278017AOOCOOOC002D003D001D002DOOOFO 2OO3730O1235OECOOOCOO2CO00CO0278E87A03COO0COO2DOO3DOO1 DO02D0O0123508E84 AEB 2OO3930O7OO3O23OC278017A00COO0COO2DO03DO01D0O2DOO01234CFCOO0COO2DO03DO01ED 20O3B300DOO2DOO012350ECO0OC0O2DOO3D0O189828B83123528COO0CO0278D27A0OCOOO7F 2OO3D30OCOO2DOO3D001D002DOOO1234CFCOO0Cω29O7O89C082CO839O7074E0F8A3EOFA94 2∞3F30(κ θ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⁾C0O2D{X)3DO01D002DO001234CFCO0OCO02DO03D001 D0O2DO0O123582 2OO51300OEC0ωCO02D003D00189828B83123528D083DO82E8F0907O89C082C0839O7O74OD 20O53300E0F8A3EOFAC000C00278087AO0COO0C0O2DOO3D0O1 D0O2D0O012348EC0O0C002B9 2005530078057 A00CO0OC0O2DOO3DO01D0O2DO0O123512C000C002C000C0O278E87AO3C0CF 200573O0O0C002D0O3D001D002D000123508E84A7O03O230C278017AOOC0O0CO02D0O3D060 2005930001DO02D0OO1234CFC0OOC0O2D0O3DO01DOO2DO001235OEC0O0C002D003DOO189C4 2005B3O0828B83123528C0OOC00278C87AωC0O0C002D003D001 D0O2DOO012352DE84A70OF 2005D300030205DAO20A16907068C082CO83907088EOF87A00C0O0CO0278017AO0C000CO46 2OO5F30ω2D003D001D002D00O123512DO83D082E8F0A3EAF09O7O68CO82C0839O7O68E018 2OO61300F8A3EOFAC0O0C0029O7089CO82CO83907O74E0F8A3EOFAC000CO0278087AO0C OBD

200633O000CO02DO03D001D002D0O012348EC00OC0O278O47A0OC00OCO02D003DOO 1 D0025B 20O65300DO00123512C0O0C002C00OC0O278E87A03COO0CO02DO03D001 D002D00012350866 2O0673O0E84A70030230C278017A00COO0CO02D003DO01D002DOOO1234CFC000C0O2DO03A9 20O693OODO01DO02D0O012350EC0O0COO2DO03D00189828B83123528COO0CO02DO03D0O1AB 2O06B3O0D002DOO0123537D083D082E8F0A3EAF0907068EOF8A3EOFAC0O0C0O278OO7A OODC

2ω6D30OCO00C002D003D001D0O2D0O012352DE84A7003020A 169O7052C082C0839 7O89A4 20O6F30OC082C083907074E0F8 A3EOFACOOOC00278087 A00C000C002D003 D001 D002D00055 20O713O01234CFCOO0C00278057A0OCOOOCOO2D003D001DO02DOO012350EC0O0C0O2COOOD9 2O07330OCO0278E87A03CO0OC(X)2DO03DO01 D002D000123508E84 A7O03O23 OC 278017 A0064 2OO753OOCOO0CO02DOO3D001D0O2D0OO1234CFC0O0C002DO03DO01D0O2D0001235OEC00 OCC

:2O077300CO02D0O3D00189828B83 I23528DO83D082E8F0A3EAF09O7060EOF8A3EOFAC0OO09 :200793O0CO02907052E0F8A3E0FAC0O0CO02D003DO01D002DO0O12354DE84A7003O207CAO9 20O7B3O0907014C082CO8378FF7AFFDO83D082E8FOA3EAF0O207DE9O7O14C082CO8378O

1AA

20O7D3O07A00DO83D082E8FOA3EAF09O7012CO82C0839O7O89C082C0839O7074E0FPES8A3E

20O7F3ωFAC0OOC⁽ 78087AOOCO00COO2D0O3D0OlD002DOOO12348EC0OOC00278O67A0O5A 2 813⁽κx θOOCOO2Dω3D∞lD 2DOM123512COO0CO02COOOC00278E87AO3C00OCOO2DO71

2008330003D001D002D000123508E84A70030230C278017AOOCOOOC002D003EX⁾01D002D08C

2ω853O0OO1234CFC0O0C002DOO3D0OlDO02DOOO12350ECOO0CO02D0O3DOO189828B831202

2OO8730O3528D083DO82E8FOA3EAFO9O7O12CO82CO83907O12EOF8A3EOFAC000COO29O7O8E

2O08930014E0F8A3E0FAC00OC002DO03D001DO02D00012348ED083D082E8F0A3EAF09O 7046

2O08B30068C082CO83907O68EOF8A3EOFAC0O0C002907012E0F8A3EOFACOO0COO2DO03

D06D

2O08D30001D0O2DO0012348ED083DO82E8F0A3EAF0907O68C082CO839O7O68EOF8A3EOF

AEA 2 8F3⁽⁾( ⁽X⁾OCω29O7O60E0F8A3E0FACOC⁾OCO02DO03D001DO02D⁽X)0123512D083DO82E8O0

20091300F0 A3 E AFO9O7068EOF8 A3 EOFAC0O0CO0278007 A00C000CO02D003D001 D002D0

005E

2O0933O0123553E84A7003020951907068C082C08378007AO0D083DO82E8F0A3EAFO907030

2OO9530O14E0F8A3E0FAC0OOCO0278O17A00C0O0CO02DO03D001 D0O2DOO012342FE84A7 0C7

20O9730O030209BC907068E0F8A3E0FAC0OOCO02907052E0F8A3E0FACOO0CO02D0O3DO018E

2OO99300D002DO0012354DE84A7003O2O9B99O7O68C082C0839O7052EOF8A3EOFADO83D0EE

2O09B30082E8FOA3EAF00209FE907O68E0F8A3E0FACOOOC002907052EOF8A3EOFACOOO

CODE 20O9D3O002D0O3DO01DO02DOOO123508E84A7003O209FE9O7O68C082CO83907052EOF8A305

2OO9F300E0FADO83D082E8FOA3EAFO907O60C082CO83907068E0F8A3E0FAD083DO82E8

F0F1

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3C3 20OA3300D001D002D00012348ED083D082E8F0A3EAFO9O7082C082CO8378017AO0DO83

D045

2OOA530O82E8F09O7062EOF8A3E0FACO0OC0O290706OE0F8A3EOFACOOOC002DOO3DO01

D045

2O0A7300O2DOO012342FC00OC0029O7O5CEOF8A3E0FACOO0C002907O6OEOF8A3EOFACO 00F2

2røA930()C0O2IXX)3DOOlD0O2DO0O123537123559CO00C0O2782O7AO0C0OOCO02DOO3DOO163

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:200AF300F8A3EOFACOOOC00278087AOOCOOOC002D003D001 D002DOOO12348ECOOOC002

785C

2røB13()OO77AOOCO0OC 2D(X33DωiD∞2DOO0123512C0⁽X⁾CO02COOOCO0278E87A03COOO7F 2ωB33O0CO02DO03DO01DOO2D0O01235O8E84A7O030230C278O17A00CO0OCO02DO03DOO199 2O0B5300DOO2D0OO1234CFCOO0CO02D003D0O1D002D00012350ECO00CO02DO03D0O1898

27D

:2OOB730O8B83123528DO83DO82E8F0120DEF1212319O7074C082CO839O7074EOF8A3E0FA43

:200B9300CO00C00278017A0OCOO0C0O2DO03D0OlD002D0OO123512D083D082E8F0A3EA

F0B2

2O0BB300907074EOF8A3E0FACO0OC0O2907089C082CO8378027AOOCOO0COO2COO0C0027859 2O0BD30OE87A03CO00CO02D0O3D001DOO2D0O0123508E84A7O03O230C278017AOOCO00CO7A 2OOBF30002D003DO01D002D0001234CFC000C002D003D001 D002D0O012350ECO00COO2

D016

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2022D30OO0C00278O17AO0C000CO02D0O3D001D002D0001235127A00D083D082E8F09O7O1E 2O22F30O84E0F87A0C⁾C⁽X⁾OCO0278137A00C000CO02DO03D00^[ DOO2D00012352DE84A7O038D 20231300022327907084C082C08378007 A00D083D082E8F0221200F A 1200FA22907087E023 20233300F87 A0OCO0OC0O278017AOOCO0OC0O2D003D001 D002D00012342FE84A700302259 A 2O23530OF1907057E0F87A00C0OOCOO278017AO0C0O0C002D0O3DO01 D0O2DOOO12342FE836 202373004 A700302239090707CC082C08378017 A00D083D082E8 F0A3EAF00225F 19070577 B 2O23930OEOF87AO0COO0C0O278027AOOC0OOC002D0O3D001DO02DOOO12342FE84A7OO3O27E 2O23B30O23B81225F49O7O57E0F87A0OCO0OC00278O37A00COO0CO02D003DOO1DO02DOOO1C 2023D30012342FE84A70030225F1907056C082C08378007AOOD083D082E8F0907064C082C8 2023F3OOCO83907089CO82CO83907O74E0F8A3E0FAC0O0C00278087A00C0O0CO02D0O3D00F 2024130001 DO02D00012348ECO0OCO0278O37A0OC0OOC002D003D001 D0O2DO00123512C0D A 2024330OOOCO02CO0OCO0278E87AO3CO00C0O2DOO3D001 DO02D0OO 123508E84 A700302307 A 20245300C278017 AOOC0O0C002DO03 D001 D002D0001234C FC00OC002D003DO01 D002DOO

OOF

2024730012350ECO0OCO02D003D0O189828B83123528COO0COO2782O7AOOC0OOCO02DOO35D 20249300DωiD002D00012348ECOOOC002780A7AOOCOOOC002D003D001 D002D0001235B79E 2024B300DO83D082E8F0A3EAFO9O7O64C082C0839O7064EOF8A3EOFAC0O0C0029O7O6A E0A1

:2024D300F8A3E0FAC000C002D003D001D002D00012348ED083D082E8F0A3EAF09O7062 COBC

:2024F3OO82C083907062E0F8A3EOFAC0OOC0O29O7064E0F8A3E0FACO0OCOO2DOO3DOOl D01C

:2025130O02D00O123512D083DO82E8F0A3EAFO907O6AE0F8A3EOFACO00C00278017AO0 C08F

202533OO0OC002D0O3D001D002DO0O12342FE84A700302259E9O7062E0F8A3EOFACO0OCO6A 202553O0O2907060E0F8A3E0FACOOOCO02DO03DO01D002DOO012352DE84A7O0302259B9O7E 2O2573OO7O62CO82C083907O60E0F8A3EOFADO83DO82E8F0A3EAFO907057C082CO8378O1ED 202593007 AO0DO83D082E8F0O225F1907062EOF8A3E0FACOOOCO029O7O6OEOF8A3EOFAC 06B :2025B30000C002D003D001D002D000123553E84A70030225F1907062C082C083907060E082 :2025D300F8A3EOFAD083D082E8FOA3EAF0907057C082C08378017AOOD083D082E8F002 35F6

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2O2673O09O7004C082C08378017AO0DO83DO82E8F0A3EAFO907O86C082CO8312361A7502ED 20269300O0D083D082E8F0907086E0F87AOOCOO0C00278007AO0COO0CO02D0O3D0O1D0O266 2026B30ODO 012366CE84A70O30226879O7O68CO82CO839O7068E0F8A3EOFAC0OOCOO29O73 2026D3OO7O86EOF87A00CO0OC0O2DOO3DO01D0O2DO0O123512DO83D082E8F0A3EAFO90 7084

:2026F30089CO82C08390707AE0F8A3EOFAC00OCO02C000COO278E87A03C0O0COO2DOO3 D0E4

2O271300O1DO02DO00123508E84A70O3023OC278017A0OC000C002D003DO01 D002DO0O124E 2O2733O034CFC0O0Cω2DOO3DO01D002D0OO12350EC0O0CO029O7O86EOF87A0OC0OOCOO28A 2027530078207AωCOXK 002D003D()01D002D0001235377A00D083D082E8F090707AC0825B 20277300C08390707AE0F8A3E0FAC0OOC0029070O4E0F8A3E0FACO0OC002D003D0O1D0 0261

:2O279300DO00123512D083D082E8FOA3EAF09O7O7AEOF8A3E0FACOOOC0O29O70O6EOF8 A331

:2O27B300E0FACO00C002DO03D001D0O2D0O09070041235ABE84A60030226879O7066C08282 :2027D300C083907068E0F8A3EOFACO00C0O278D27A0OC0O0CO02DOO3D001 D002D00012 3591

:2027F300B7D083DO82E8F0A3EAF09O7066C082C083907066E0F8A3E0FAC0O0C00278D2 7A29

:2O2813000OCO0OCO02D 03DO01D0O2D0O012348ED083D082E8FOA3EAFO907O68CO82CO8322 :202833009O7O68E0F8A3E0FACO00C0O29O7066EOF8A3E0FACOO0COO2D0O3D001DO02D0 00C3

20285300123537DO83D082E8F0A3EAF09O7086C082C08312361 A75020OD083D082E8FO9OFC 202873007086E0F87A00C000C00278007AO0C000C0O2DO03D001 D002D00012366CE84A706B 202893O003O2285F9O7086CO82CO839O7O86EOF87AOOCO00C 00278207 A00C000C002D003CD 2028B300DO01D0O2DOO01235377AO0DO83D082E8F0907086EOF87AO0C0OOC002907O68E07B 2028D300F8 A3 EOF AC000C002DOO3DO01 D002D00012366C E84 A70030228 FBD2179037B7 12B 1 :2028F30O35881233D8123416907O68E0F8A3E0FACO00C0029O7O86EOF87A00CO00C0O2D026 2029130003D001D002D00012342FE84A7003022637D2179037B91235881233D81234169074 202933O07O78C082CO8378017A00D083DO82E8F0A3EAF0907068C082C08378007AOOD083F8 202953O0D082E8F0A3EAF0907O7ACO82CO8378007AOOD083D082E8FOA3EAFO9O7O02CO 828E 2O2973O0C08378O77A0ODO83D082E8F0A3EAF0907000C082CO8378017AO0D083D082E8F019

2O2993O0A3EAF0907086C082C08312361A750200D083D082E8F0907086EOF87A00COO0C0EE 2O29B3O00278OO7A00C0O0COO2DOO3DO01D0O2DO0O12366CE84A7OO3O229969O7O68C08284 2029D3O0CO83907O68EOF8A3EOFAC000C002907086EOr87AO0COOOCO02D003DOOlDO02 D0C2 2O29F30O00123512D083DO82E8F0A3EAFO9O7089C082C083907078EOb8A3EOFACOOOCO0214

202 A 130078087 A00COO0C0O2DOO3DO01 D002D00012348 ECOOOC00290707 AE0F8 A3E0FA COFC

2O2A3300O0C0O2DO03D001D0O2D000123512C0O0C0O2CO0OC00278b87Λ03C0O0C002D003EC 2O2A53O0D001D0O2DO0O123508E84A70O3O230C278017A00C0O0C0O2DO03D001 D002D0004D 202A73 i234CFCO()OCO02D003DO01DOO2DOOO12350bCOOOC0(⁾29O7086EOF87A0OCOOOCO37

2O2A93O0O2782O7AOOCO0OCω2D0O3D0OlD002D0001235377AOOD083DO82E8F090707AC098 202AB30082C08390707AE0F8A3E0FAC0OOC0029O70O0E0r8A b0I ACO OC002D003D001 D0A2

202AD30OO2DOO0123512D083DO82E8F0A3EAF09O707Ab0F8AirorACO00C0029O7O02EO F893

202AF3O0A3EOFAC0OOC002D003DO01DOO2D0009070001235ABF84A6O03O229969O7O66CO10 202B 130082C083907068EOF8 A3 E0FAC00OCOO278D27 A0⁽r0OOCO02DO03 D001 D002D0O01200 202B330035B7D083D082E8F0A3EAF09O7O66C082C0839O7066C01 8A3E0FACO0OC 00278 D22A 2O2B53OO7AOOCOO0C002DO03DO01D002D0OO12348ED08 ^:SDϋ82F8FOA3EAFO9O7O68C082

C0E8

202B730O839O7068E0F8A3E0FAC0O0C002907O66EOF8A3l θr ACO0OC002D003DOOlDO02 DOFD

202B930000123537D083D082E8FOA3EAF0907086C082C08 1 361 A750200D083D082E8F049 2O2BB3O0907O86E0F87AO0CO0OCO0278007AO0CO0OC002DOO3DO01 D002 DOOO 12366C E84A08

202BD3O07O03O22BA0907086C082C083907086E0F87AO0C00OCO02782O7A00C00OC0O2D0D9

202BF30003DO01DOO2DO001235377AO0D083D082E8FO9O7O68E0F8A3EOrACOO0COO29O7O98

202C 130086E0F87AO0CO00CO02D0O3D001 D002D00012342FF84A7003022C6ED2179037BBE0

2O2C33001235881233D81234169O7078C082C083907O78E0F8A3E0FACOO0CO0278O17A00FA 202C5300COOOC002D003D001D002D000123512D083D082b8FOA3EAr0022C7CD2179037

BD2F

2O2C73O01235881233D8123416907O78E0F8A3E0FAC000CO029O7O89CO82C08378027A0OA8 : 2O2C93OOC0OOC002COOOC00278E87A03C0OOCO02D003DO01 D002DOOO 123508E84 A70030282 :202CB3OO30C278017AOOC0O0CO02D0O3D001D002D0001234CFC000C002DO03DO01DO02 D077

202CD3000012350ECO0OC002DO03D00189828B83123528CO00COO2D0O3D001 D002DOOO 1204 2O2CF3O0352DE84A7003022946907087C082C08378017AO0D083D082E8F09O7086C082CO45 2O2D13OO8378O07AO0DO83D082E8F0907O86EOF87A00C0O0CO0278097AO0COO0CO02DOO3O4 2O2D33OOD001D002DOO012342FE84A7003022D5790707ECO82C08378017A00DO83D082E8EA 202D53OOF0A3EAFO9O7086E0F87AOOCOOOC0O2780A7AO0COO0C0O2D0O3D0O1D0O2DOOO 12C3

2O2D730O342FE84A7003022DDF907087C082C083907O87EOF87AOOC000COO278017AOOCO1O 202D93OOOOC002D003D001D002D0001235127A00DO83D082E8F09O7O87E0F87A0OC000C06F 202DB30OO278O17A0OCOO0C002D0O3D001DO02D00012352DE84A7003022DDF9O7087C08253 2O2DD3()OC08378TO7AWDO83D082E8F09O7086EOF87AOOCOOOCOO2780B7A⁽⁾OCO00CO02DO85 2O2DF3OOO3D001DO02D0O012342FE84A7O03O22E15907086C082C08378O07AOODO83DO8249 202E 1300E8F0907086E0F87 AO0CO0OC0O2780C7 A00C0O0C002D0O3DO01 D002D00012342F32 2O2E33O0E84A7O03O22F03907086CO82C08312361A750200D083DO82E8F0907086E0F87A6D 202E53CW()0C(X)OC00278007A(⁾0CωθC 2DO03DO01DO02D0O012366CE84A7OO3022E3A9OD0 2O2E730O7086C082C083907086E0F87A0OCO0OC00278207AOOCO00C0O2D003DOO1 DO02DO9O 202E9300O01235377A0OD083D082E8FO907087E0F87A00COOOC00278007A00C000C002D00B 202EB3OOO3D0O1DO02DO0012342FE84A70O3O22EF2907086EOF87AOOCOO0COO278017AOOO0 202ED300COOOC002D003D001D002D00012342FE84A7003022EEF120153022EF2120156905D 202EF3OO7086C082C08378007AO0D083D082E8FO907086EOF87AO0CO00C00278OD7A0OCOBC 2O2F13OOOOC0O2D003D0O1D002DO0012342FE84A7O03O22FF7907086C082C08312361 A7572 202F33OO02O0D083D082E8FO907O86E0F87A00C0O0C00278O07AOOC0O0CO02DO03D0O1DOBD 202F530002D00012366CE84A7003022F28907087EOF87AOOCOOOC00278007AOOCOOOC0020B 202F7300D003D001DO02D00012342FE84A7O03022FE6907055C082CO83907086EOF87AOO15 202F9300C0O0C00278207A00C00OC0O2D0O3DO01D002DOO01235377A00D083DO82E8FO9OBD 202FB3007055EOF87AOOCOOOC00278007AOOCOOOC002Dω3D001D002D00012342FE84A7094 2O2FD3O003022FDD12O159022FE612015A12O148120156907O86CO82CO83780O7A00DO83C9 202FF3 OD082E8F0907O86E0F87 AOOCOOOC002780E7 A0OC0O0C0O2DO03 D001 DO02DOO01260 203013OO342FE84A700302302D907O86CO82C08378007A0OD083DO82E8FO907086C082CO34 203033008378007 AO0D083D082E8FO22O236728587E02OE5FADOD0D0EODOE0D2D574AA 759A

:2O3053OOF0559O0033C082C0833253883F53892F43892O43878O888D758B00D28E2275C77O :20307300AA75C755D2BF22I2307175C7AA75C755438704D21D2275C7AA75C7555387FB C238 :203093001D2289828B83E493C082C08388828A83F06017DC09E4F0158115810230CAA3A81 F :2030B30082AA83D083D082A380DC 15811581229037BF74460237399037D374500237398852

: 2030D300298 A2A2230191 C301705D21 AD0D0222008FDC082C08390748BC2D5D21 BC21 A

C026

:2O30F300DO8O0ED0D032C082C083907489D2D5C0DOC0EOCOFOCO00CO01C0O2C0O3CO04

C06A

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

3F81A

:2031F300EA33FA22EC33FCED33FD229O37F374140237399037E4740A023739903810742866

:2032^]30O0237395389F0D21O43A882D28C22907489E8F0A3EAF043A88222E859F822E84991

:2O323300F82290748BE8F0A3EAF0D219D2BCC2BAD2B943A884229O74F87475F0A374OO

F090

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

2034F3000231FEE4C398F8E49AFAC322E4C399F9E49BFBC322123476O23467D2D58002C218 20351300D5C3E829F8EA3BFA10D504A2D2C2D250030231FE22E0F87A0022123435023466B6 20353300D2D58002C2D5C3E899F8EA9BFA10D504A2D2C2D250030231FE221234760234 660E

20355300123435023467EA30E70A1234F6EA30E70302320E2212343502346CE859F8EA5BF6 20357300FA22E849F8EA4B FA2212347C02346612343502346BE49360061233 A4 A380F62029 203593O0O8FD22123476023462E52AA829B52AF9FA22EOF8A3E0FA22C3A3EO33400302355F 2035B30068023582EB33EA1234599035C 17380118013801E 1234F61234FF1231AA1234FFC2 2035D300221231AA221234F61231AA1234F61234FF221234FF1231AA1234F622D007D0069E 2O35F300DOO5DOO4DO03D002DO01DO0OD0F0D0E0DOD020D509DO83D082D0DOC21B32 D08314

:20361300D082DODOC21322COA853A8EB9074A1EOF890749DAB8290749EEOF9A3EOB500 045E

20363300780O801A8983F582A3E582B5O3039O748DE0F8AB82E5839O749EFOEBA3F0DOA897 2036530O229O748DAA83A98290749EEAFOA3E9F09074A0EAF0A3E9F022123476O23460C224 20367300111233A080FE7433C33O1B02940EF5F0E581 B515004O0C747FF425814O0525F022 2036930040012285158102369C9038717464023739C082C0831233D890389712358812362A 2036B3O0BFD083D0821235881233D82290389E12358890755B 1235A5907551 A982AB831243 2036D30036E29075511233939038A512358822C3E84A7006COE07C01800E7C00790A1237D5 2036F30007C0010CE84A70F47930DOE029FOA3DCF9E4F022AF02AEOOAB0178077902EF 8BEE

:20371300F084AAFOFF18E6D7C48BF084AAFOC4C6C4D7C48BF084AAFOD6A806AA07A 9F022E5

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

203893004C4544004552524F522000284C696E652000293A2000535441525455502F52454A

0438B3005345540025

00003301CC

Stepper processor(IC 3) hex file(Step23.hex):

O30OOOOO020O33C8 05O0OB00C0D0021 EB68A 050013OOCODO021 E68D0 05002300COD0021FDF48 O5OO2B0OCODOO21C3CE6

20003300C2AFC2D4C2D3752100752200752300D215D28AAF 17581 08581 15C0D0C0F0C098 2OOO53O0E0C08753C6FB121C837527405388CF5389F0121 FOE78FC 1 1 C57121 F6E1221AC99 20007300C20EC20FC208759850D2ACD2AFE587DOEODOOODOOI DOD030E20530E4028012 206A

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20177300F AC000C0O278407 A 1 FCOOOC002D003D O 1 D002D000121 D59E84 A70030217C 412 A5 2O1793O0O631907010C082CO83788O7A3ECOOOCOO290701OE0F8A3EOFACOO0CO02D0O3D0AE 2O17B300O1D002DOO0121D8ED083D082E8F0A3EAF0907015CO82CO8378OA7AO0DO83DO

8281

:2017D3O0E8F012O14E907OOEE0F8A3EOFACO0OC00278FF7AFFCO00CO02D0O3D0OlDOO2

D020

2017F3O00O121E5DE84A7O030218O1 12O6319O7019C082CO8378017A00D083D082E8FOO23O 201813001823D2179023671221C41220FD12213B907017C082C08378007AOOD083D082E8C8 20183300F0907017EOF87AO0C0OOCO0278087AO0CO00C002D003D00I D002DO00121E5DE883 201853O 4A70O3O2186A907017C082CO8378OO7AO0D083D082E8F09 7O17EOF87A0OC 00000 20187300C002780A7AOOCOOOC002D003D001D002D000121E5DE84A70030218A0907017COOC 2018930O82C08378OO7A00D083D082E8F09O7017E0F87A0OCO0OC002780C7AO0CO0OCOO296 2018B300D003D001D002D000121E5DE84A70030218D6907017C082C08378007AOOD083D

OFC

2018D30082E8F0907017EOF87 A0OCOOOCOO2780D7 A0OC0OOC0O2D0O3DOO 1 D002DOO0121 EB9 2018F3005DE84A7003021972907017C082C083122172750200D083D082E8F0907017E0F822 201913007 A00C0O0CO0278O07A00C00OC0O2D003D001D0O2DO00122154E84A7O030218FBBD 20193300907017EOF87A00C00OC00278217A00CO0OC002DO03DO01 D002DOO0121E5DE84AOF 20195300700302195E12068702196112068A907017C082C08378007AOOD083D082E8F09030 201973O07017E0F87A00C0O0CO0278OE7 AOOCOO0C002D003DOO 1 D002D000121 E5D E84A7002 201993O0030219A89O7017CO82C08378O07AO0DO83D082E8F09O7O17CO82CO8378O07AO0D5 2019B300D083DO82E8F022907016E0F87A0OCO00C00278007A0OC0OOCO02D0O3D001 DOO2A1 2019D300D000121E5DE84A7O03021A1D907018E0F87A00CO0OC0 2780O7A00COO0C0O2D089 2019F30003D001D002D000121D59E84A7003021A1AD2A4907016C082C08378017AOOD083A4 201 Al 300D082E8F0021 A 1 D 121 AF30221 EC78007 A00121 F2E 121 F20C000C002907009E0F81 D 201A33007AO0C0O0C002D003DO01DO02D0O0122217E84A7O03021A27D2A378O07A00121 F86 201A53002E907006C082C083A8A07AOOCOOOC00278207AOOCOOOC002D003D001D002DOO

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D28

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201BD30000121D8AD083D082E8FOA3EAF0907010C082C083907010FOb8A3EOFACOOOCO 02C3

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:201D53005003021D2722121D5F021CD8C2D5EB33EA121D83901D6B738012D3222200EA

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AC325

:201DF30022E4C399F9E49BFBC322121D5F021CDDC3E94B7005D3021 D2F227F007E007C

0065 :201E13007D0075F010121E48EE33FEEF33FFC3EF9B401 17005C3EE99400AC3EE99FEEF

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:201E3300FFD38001C3121E50D5FODA8C008D028F038E01C322C3E833F8EA33FA22EC33

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:201E5300ED33FD22121CE8021CD8121 D5F021CD488298A2A2230191C301705D21ADOD 022BE

:201 E73002008FDC082C083907071C2D5D21BC21 ACOD0800EDOD032C082C08390706FD2

D549

:2Ol E9300C0D0C0E0C0FOC000C001C0O2C003C0O4CO05CO06CO07D215E0F8A3E0F58385

00F4 :201EB30082E473301052COE085278CE52804B4C808E40529B52902052AF528301 13820134D

:201ED3O0352O12OEE52BB52A07E52CB529O28002502420171 F2O081CE5C620E217DOE0D2BD

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7116

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:201F9300F8907ODCE0F9A3EOF5828983E8F0A3E582B45O03907100A882E5829070DDFO88AB

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: 2021 F30004D003D0O2DO01 DOO0DOFODOEOD0D020D509D083 D082D0D0C21 B32D083D08 2D0AB

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:2022B30002AEOOAB0178077902EF8BF084AAFOFF18E6D7C48BF084AAFOC4C6C4D7C48 BF098

:2022D30084AAF0D6A806AAO7A9F022E8FOA3EAF022B42800500122COE0C0O0C0O2COE 0C095

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20243300636174696F6E204641494C4544004552524F522000284C696E652000293A200049

0E2453OO535441525455502F52455345540O96

00003301CC

Serial snoop processor(IC 16) hex file(Serial5.hex):

10000000680006061EOA1209080C2A0003042803C9 100010000606E8051009EA02060A08022900000 A95 10002000540C 130 A6C0C2BOOEBO2140 A000801 OC 90 100030000600000C0500060C2500000A 120C8901 CO 1000400043066504130C890143066505140C8901 F8 1000500043060504150C890143060505160C8901 A4 1000600043062504170C890143062505180C890150 1000700043064504190C890143064505200C2B0055 06008000EB02400AOOOA39 0203 FEOO 170 ADC 0000000 IFF