Process for validating thermocyclers

The invention relates to a method for the validation or referencing of thermal cyclers, in particular in PCR devices, the method comprising at least two reactions in which the annealing temperature and the denaturation temperature, for example of a sequencing reaction or amplification reaction, are checked; the invention also relates to a kit and its use for the validation of thermal cyclers.

The polymerase chain reaction (PCR) was developed by Kary Banks Mullis in the early 1980s. His idea was to develop a process that artificially multiplies DNA by repeated duplication in several cycles using an enzyme called DNA polymerase. DNA polymerase occurs in all living things, it doubles the DNA before cell division. It binds to a single strand of DNA and creates a complementary strand. Mullis' original PCR experiment used the enzyme in vitro (in a controlled, artificial environment). The double-stranded DNA was divided into two single strands by heating to 96 ° C. At this temperature the DNA polymerase was destroyed and therefore had to be renewed after each heating. Mullis' original process was very inefficient because it required a lot of time, large amounts of DNA polymerase and constant attention. The PCR process was later improved by using DNA polymerase from thermophilic bacteria living in geysers at over 110 ° C. The DNA polymerase of these living things is and was therefore not destroyed when heated during the PCR cycles. Since it was no longer necessary to continuously add new DNA polymerase, the duplication process could be considerably simplified and automated. One of the first thermostable DNA polymerases was made<i>Thermophilus aquaticus</i> won and called Taq. Taq polymerase is currently widely used. A disadvantage of Taq polymerase is that it sometimes produces errors when copying the DNA, which leads to mutations (errors) in the DNA sequence. Polymerases such as Pwo or Pfu, which are obtained from Archaea, have a correction mechanism that significantly reduces the number of mutations in the copied DNA. But numerous chemical reactions, such as sequencing reactions or amplification reactions, only achieve their optimal efficiency and effectiveness if a certain defined temperature regime is observed.

With the establishment of biochemical processes in the laboratory routines of basic research and medical clinics, it had become common to carry out numerous in vitro reactions, for example in a water bath. If several reaction batches, for example in test tubes or plastic vessels, such as tubes, are placed in the identical water bath at the same time, it is ensured that all reaction batches are brought into contact with the same ambient temperature.

For example, when Kary Mullis invented PCR (Polymerase Chain Reaction), he used various water baths in which he positioned his respective reaction approaches. With the diagnostic use of laboratory routines and the increase in parallel examinations, it is no longer possible to temper the reaction batches with the aid of water baths without further automation. Furthermore, only one temperature can generally be kept constant within one and the same water bath; it is not possible to bring the reaction batches into contact with different temperatures, temperature intervals or temperature profiles in a water bath within a short time or within defined time intervals.

For this reason, alternatives were sought which, on the one hand, enable a homogeneous temperature control of several reaction batches as well as the change of different temperatures, i.e. the coordination of specific temperature and time intervals. This concept was realized with the help of thermal cyclers.

Thermal cyclers are periodically heating devices that are provided in different versions, for example the devices can be heated and cooled with the help of liquids, or they can heat up via an electrical resistor and be cooled with semiconductors (Peltier). As a rule, the thermal cyclers have a metal block in which recesses or holes are made, in which the reaction mixture, for example a tube, can be positioned.

Both the PCR and the sequencing of nucleic acid or amino acid structures are young, but already established methods, the further development of which requires increasingly precise and effective thermal cyclers. In the meantime, these processes have also achieved a permanent position as a diagnostic method in human medicine, veterinary medicine, the food industry, biotechnology and pharmaceutical research, also as a result of the further improvement in the performance of the thermal cycler.

In the PCR reaction, the method is essentially defined via the primer sequence and a PCR protocol with specific temperature / time intervals. PCR methods are usually developed in such a way that slight deviations of the individual steps of the actual values ​​of the temperature program of the thermal cycler from the target values ​​exert as little influence as possible on the result of the PCR reaction. The robustness of the reaction achieved is different for each PCR method. With each method, however, the detection limit, i.e. the sensitivity or the selectivity, is impaired in the event of a deviation from the target values ​​which is dependent on the respective method. The proof is therefore dependent on the correct temperature control of the thermal cycler used. To avoid false positives or false negative results, it is essential to check the correct temperature control of the devices used.

In particular when using thermal cyclers in PCR, negative and possibly also positive controls should run. Despite the uniformity of the metal block, a homogeneous temperature distribution and thus identical results with identical reaction batches are rarely achieved. Theoretically, a metal block should have the same temperature everywhere. In practice, however, the temperature can differ noticeably from one reaction site to another, so the cycler parameters fluctuate accordingly and, as a result, the results achieved. Furthermore, the temperature and time intervals of a metal block also vary, especially when the thermal cycler is used for a long time, which means that it is no longer guaranteed that the specified device-specific temperatures will be reached correctly in the specified time intervals.

Especially when using so-called multiblock systems for large users with high throughput, a homogeneous, constant temperature distribution (i) within the heating blocks and (ii) within the time intervals as the absolute temperature reached at a defined location, especially after long use (e.g. years) of the device usually cannot be guaranteed.

Almost all devices currently have a standardized block for holding PCR tubes. The temperature control of the devices is not standardized and differs considerably depending on the manufacturer of the thermal cycler and model. Due to the use of Peltier elements, all devices also show a performance-dependent decrease in performance in the precise temperature control, a consequence of the aging of the Peltier elements. Correct temperature maintenance is crucial for the specificity and sensitivity of a PCR detection.

The temperature distribution on the heating block and the temperature control are checked, for example, by measurement using physical-technical measuring methods. To do this, it is necessary to send in the respective thermal cycler, which means that it cannot be checked during ongoing operation in the laboratory as part of quality management. The spatial design of the measuring probes disadvantageously creates thermal conditions in the thermal cycler that differ from reality, which essentially means that the results differ from the laboratory reality.

Another method in the prior art is measurement using non-standardized PCR methods within the laboratory. The disadvantage here, however, is that all the PCR methods used for the routine are more or less very robust systems, which means that changes can only be detected when this already very robust system is disturbed. In particular, it is not possible to say how the robustness of the method used relates to all other PCR methods in the laboratory. From this it can be concluded that it should actually be checked regularly against all other laboratory methods, but this is practically impossible. Furthermore, no separate statements can be made about the problems with denaturing and / or annealing.

The invention is accordingly based on the object of providing a method which avoids the disadvantages mentioned, which result in particular from measurement using physical-technical measurement methods or measurement using non-standardized PCR methods within the laboratory, the method being designed in this way should enable simple, safe and efficient validation and referencing of thermal cyclers.

The invention solves this technical problem by providing a method for the validation or referencing of thermal cyclers, wherein at least one PCR reaction batch is positioned in the thermal cycler or is operatively connected to it and at least one test of the annealing temperature and the denaturing temperature is carried out.

The invention thus relates to the surprising teaching that by checking two temperatures, namely the annealing temperature and the denaturing temperature, a referencing of thermal cyclers is possible without the known disadvantages. By determining two temperatures, it is possible to validate thermal cyclers without the measurement using physical-technical measuring methods, such as measuring probes. Furthermore, separate statements about the problems with denaturing and / or annealing are possible, the method according to the invention advantageously being able to be checked regularly and easily against all other methods in the laboratory.

The method according to the invention accordingly comprises two PCR reactions, which in particular take place in a vessel, the first PCR method checking the annealing phase and the second PCR method checking the denaturation phase. In particular, the methods use a different primer and form products of different lengths and / or sequence sequences. The method is designed so that the PCR methods are particularly sensitive to temperature.

In the validation of the thermal cycler according to the invention, it is in particular possible for a certain number of reaction batches to be distributed over the surface of the heating block of a thermal cycler - according to a random random pattern, arbitrarily and / or in an ordered manner. The positioning depends on the thermal cycler to be examined. A thermal cycler can have, for example, 5, 10, 24, 96, 384 or more cavities.

A polymerase chain reaction in the sense of the invention is any method for the propagation of DNA. It is preferably a thermocyclic reaction, with the amount of DNA provided at the beginning of the cycle being theoretically doubled in each cycle. For the purposes of the invention, annealing means the attachment of the primer to the DNA template. After the entire DNA has melted, the reaction mixture is cooled to a very precisely defined temperature. At this so-called annealing temperature, the primer binds specifically to its template. However, also for the renaturation of the original double strand and for non-specific attachment. Renaturation is prevented, for example, by the large excess of primer. Only after binding the primers does the rest of the template renaturate without displacing the primer. The non-specific accumulation is avoided by the annealing temperature being only very weak (approx. 2 °) below the melting temperature. Naturally, the specific binding is more stable. This is exploited: at their stable binding sites, the primers bind just enough, at the non-specific binding sites, however, there is a continuous melting. In critical cases, the annealing temperature can be chosen higher than the melting point. This reduces the yield, but increases the specificity. Because the base sequence of the primers is known, you can always calculate your melting temperature and thus also the annealing temperature. Including all possible factors - mainly the purine-pyrimidine distribution - the melting temperature can be determined with accuracy down to the decimal place. As a rule, the application of the Wallace rule is sufficient. This includes separately adenosines and thymidines or guanosines and cytidines. The AT value is multiplied by 2 and the GC value by 4. The addition gives the melting temperature in ° C. Typical primers give values ​​between 55 and 65 ° C. The melting temperature should be as similar as possible for both primers. If the two temperatures differ, the lower temperature must be used. This leads to an increase in non-specificity in the counterprimer. The concentration of primer decreases sharply in the course of the reaction. In order to achieve an optimal bond in this area as well, the annealing time can be varied. If the DNA template is only in a very low concentration or if the reaction mixture is not clean, eg contaminated by strong foreign ions, an extension of the annealing time can be helpful so that the primer can find its template.

For the purposes of the invention, denaturing means the strand separation. The mechanisms of strand separation depend on the architecture and stability of the double helix. The stacking forces are based on interactions between the Π-electron systems of the quasi aromatic bases. The effect is also known as a charge transfer complex. From a length of about 30 nucleotides, the stability of the double helix is ​​no longer dependent on the length of the polymer. A strictly cooperative behavior can be demonstrated. This applies to a statistical distribution of the nucleotides. GC-rich sections, on the other hand, show increased stability compared to AT-rich sections. Other factors that affect stability can be the superstructure and associated proteins. Special conditions exist for oligonucleotides (up to approximately 30 bases) and at the end. Here the stability of the helix is ​​still dependent on the length of the DNA or depending on the distance to the end; the ends are the least stable. The terminal nucleotide can only generate stacking forces in one direction. As a result, it is almost always free even at low temperatures. The second nucleotide is also relatively free if the first nucleotide does not develop any stacking forces. In this way, stability gradients can be observed. The effect can only be neglected from nucleotide four. This in turn means that an octa-oligonucleotide (8-mer) is the first oligomer with measurable stability, provided the sequence is self-complementary. This double helix is ​​no longer stable even at room temperature.

In a preferred embodiment of the invention, the validation takes place in connection with a real-time PCR. Real-time PCR measures the origin of the PCR products during amplification. The method is based on changes in fluorescence that are measured through the transparent vessel walls or the lid. Fluorescence is the property of substances to release absorbed energy as a light quantum of lower energy. In recent years, real-time PCR devices have come onto the market with which various real-time PCR methods can be used. The simplest real-time PCR method is based on the addition of SYBR Green, a dye that increases its fluorescence when attached to the double-stranded PCR products. Contrary to the classic method of detection in agarose gel, the additional information about the length of the product is lost; the method is therefore not suitable for verifying the PCR experiment. A subsequent melt analysis can be used to make a rough estimate; at least primer dimers, which are frequently present by-products, can be identified in the melt analysis. All other methods use fluorescent labeled primers or probes.

The oldest and probably the most widely used method is the 5-nuclease assay, better known as the TaqMan assay. A double-labeled oligonucleotide probe is used, which carries a reporter fluorescent dye and a quencher. The quencher quenches or reduces the reporter's fluorescence by collision of the probe ends, or by a fluorescence resonance energy transfer (FRET). The probe binds to the resulting PCR product and is hydrolyzed by the polymerase, so that reporter and quencher are separated, which increases the fluorescence. This signal is therefore dependent on the existence of the binding site and verifies the PCR reaction.

A touchdown PCR is also preferred, in which the specificity of the primer binding is increased by cyclically approximating the annealing temperature to Tm. With the touchdown PCR, the annealing temperature is approximated to the expected melting temperature of the primer (TM) in cycles and lowered further. If the annealing temperature is chosen above the expected Tm, the primers bind exclusively and therefore highly specifically to the DNA. Primer dimers and artifacts are reduced, and the desired amplicon preferably multiplies.

A prolongation PCR is also preferred, in which the allelic dropout effect is to be avoided by extending the elongation phase. Prolongation PCR is a method in which the last step of each cycle is extended. This is to prevent the "allelic-drop-outeffect". One wants to prevent the termination of the synthesis before reaching the end product. If one extends the elongation phase in proportion to the decrease in active enzymes, templates and dNTPs, the end point is always reached and all copies of the gene have the same chance of being amplified. The synthesis gains in efficiency and longer DNA fragments are also completed. This method is mainly used for heterogeneous DNA mixtures. It is primarily used when two polymerases are used at the same time, such as in the ROCHE EXPAND-PCR system. One polymerase ensures a quick synthesis and the other for quality control by means of "proofreading". Allelicdrop-out-effect: In a heterogeneous DNA mixture, such as in phorensic determinations, certain alleles are represented with different frequencies. Since the PCR amplifies exponentially, this unequal distribution can be amplified so much that the less concentrated allele is so poorly represented that it can no longer be detected.

A hot start PCR is also preferred, in which it only starts the polymerization when a minimum temperature is reached. The hot start or hot start PCR differs from the general procedure only in the first step. In this case, the polymerase chain reaction is only started when the reaction mixture has reached the desired maximum temperature. This means that the polymerization only begins when the primers have specifically bound to the DNA sequence. You get fewer artifacts. This can be achieved by different methods. The original variation was the addition of the polymerase only after the melting temperature of the DNA had been reached. This process has several disadvantages. Because the polymerase is pipetted into many samples in succession by hand, the reactions are no longer started at the same time. In addition, contamination can result from opening the reaction vessel. Another method is to separate the reaction components using a wax layer. First, the templates, buffers, dNTPs, primers and supplime are added to the reaction vessel. Then add a wax bead of a defined composition (commercially available). When heated for the first time, the wax melts and seals the incomplete reaction mixture. Then the polymerase is added. If the wax melts when heated and rises, the aqueous phases mix. The advantage of this method is that all reaction batches are started at the same time. Due to the defined chemical properties of the wax, the experiments are easily reproducible. Third, there is the possibility of delayed start of the reaction by using antibodies. In this case a DNA polymerase is used which is bound to antibodies. The antibodies block polymerase activity at normal ambient temperature. Since the antibodies are not thermostable, they denature and dissociate when heated for the first time. The polymerase can now begin its work. This is a specific method since the polymerase is only active when the temperature is so high that the primers bind specifically to the DNA due to the higher temperature. The advantages of the hot start method are: fewer or no artifacts accumulate; the polymerase reaction only begins when the DNA has melted completely; the polymerization only begins when the primers bind specifically to the DNA due to the temperature, and the primer concentration is not reduced by primer dimerization.

In the likewise preferred nested PCR, the first PCR product is used as a template for a second PCR after a successful first amplification. The nested PCR is a method to further increase the specificity of the PCR. In a first step, the PCR is carried out with a template over a few cycles. Then new primers are used in a second round. The new primers are located further inside than the first ones, so that in this second step only the specific DNA sections of the first step are amplified. It is also possible to use only a primer located further inside. This increases sensitivity and efficiency so that work has to be carried out very carefully and contamination-free, since contamination can have undesirable effects.

The likewise preferred RT-PCR serves for the detection of RNA or a double-stranded DNA which is complementary to the RNA. This method can be used to study expression patterns at the mRNA level. In addition, highly sensitive viral RNA can be detected by RT-PCR. The RT-PCR is divided into two steps. In the first step, the RNA is transcribed into cDNA ( c stands for complementary) by reverse transcription . This is necessary because many polymerases are unable to use RNA as a template. The second step is followed by a normal PCR in which the cDNA generated in the first step is used as a template. It is possible to separate these two steps spatially and / or temporally. The conventional method is to first add reverse transcriptase to the reaction mixture and then use it as a template in the PCR approach. Another method is to use Tth polymerase (from Thermus thermophilus). Tth polymerase can work both as a reverse transcriptase and as a polymerase. The regulation is carried out by certain cofactors: in the presence of manganese ions, the Tth polymerase shows a reverse transcriptase activity, and in the presence of magnesium ions a DNA polymerase activity. By adding the right cofactors, the entire RT-PCR can be carried out in one batch.

In a further preferred embodiment of the invention, the annealing and denaturation temperatures are checked in at least two reaction batches. The annealing temperature is advantageously checked in one reaction batch and the denaturation temperature in a second reaction batch. Advantageously, the components that are used for the determination in the reaction batch do not interact with one another.

In a further preferred embodiment of the invention, a DNA template is used to check the temperature, the DNA template being characterized by a high T_M-Value of at least one section is temperature sensitive. It is advantageous because of the selection of the high T_M-Value section of the DNA template possible to check and reference the denaturation temperature.

In a further preferred embodiment of the invention, at least one primer is used to check the temperature, in particular the annealing temperature, the primer being sensitive by a mismatch in such a way that the annealing temperature can be determined. The temperature sensitivity of the PCR is advantageously achieved via at least one mismatch, in particular at the 3 'end of at least one primer, which makes it possible to check the annealing phase of the PCR reaction. The one or more mismatches at the 3 'end of a primer advantageously result in a reduction in effectiveness, as a result of which the PCR reaction, in contrast to the usual reactions, is not very stable and robust and is therefore very sensitive to temperature deviations.

The primer having at least one mismatch at the 3 'end can be used in particular in a master mix. The master mix preferably contains all the components which are required for carrying out a PCR; preferably PCR buffer, MgCl₂, dNTP's and / or Taq polymerase. The master mix furthermore contains the necessary probes known to the person skilled in the art for carrying out real-time detection.

In a further preferred embodiment of the invention, the primer comprises the following sequences in particular when checking the annealing temperature:<img file="EP1512757A2_D0001.tif" /> and<img file="EP1512757A2_D0002.tif" />

In a further preferred embodiment of the invention, the annealing temperature in the PCR mixture with a target DNA with the sequence<img file="EP1512757A2_D0003.tif" /> checked.

In a particularly preferred embodiment of the invention, the denaturation temperature in the PCR reaction mixture is checked by using a high-melting PCR template, the T_M at least one section in the template is greater than 90 ° C.

Various possibilities are known to the person skilled in the art, the T_M- Increase the value of the section in a template, in particular it is possible to select the respective section, especially GC-Reich.

In a further preferred embodiment, the template preferably has the following sequence for determining the denaturation temperature:<img file="EP1512757A2_D0004.tif" />

In a further preferred embodiment of the invention, the denaturation temperature in the PCR reaction mixture is checked in particular by using primers with the following sequences:<img file="EP1512757A2_D0005.tif" /> and<img file="EP1512757A2_D0006.tif" />

In a particularly preferred embodiment of the invention, a defined amount of two different target DNAs is used in a PCR reaction mixture. For example, it is possible that the reaction batches contain, for example, 10,000, 1,000 or 100 copies of the starting or target DNA. Through routine tests, the test can advantageously be set so that, with a correctly working temperature cycler, all reactions with 10,000 and 1,000 copies generate sufficient PCR products which, for example, can be clearly detected as a band in a subsequent gel electrophoretic evaluation. Of course, the defined quantities can be selected in almost any area. For example, 20, 200, 2,000, 20,000 and 200,000 or 50, 500 and 5,000 copies can also be used, which can be compared after the annealing temperature and denaturation temperature have been checked. It is preferred that 2 or 3, but also 4, 5, 6 or even more different defined amounts of both of the respective target DNAs are used.

It is further preferred that the checking of the annealing temperature and the checking of the denaturing temperature are carried out as a multiplex reaction. This means that all primers suitable for the respective reactions are advantageously used at the same time.

In a further preferred embodiment of the invention, the checking of the annealing temperature and the detanuring temperature is carried out by means of separate evaluation of PCR products of different sizes and / or sequences. It is particularly possible to examine the products in the gel for PCR products of different sizes. If real-time methods are used, it is advantageously possible to examine the different sequences. In classic PCR, it is preferred to bring at least both products from a multiplex PCR into a gel pocket, and in real-time PCR it is preferred to carry out a multiplex-PCR with real-time probes.

In a particularly preferred embodiment of the invention, 12 to 17 PCR reaction batches are used. The number of PCR reaction batches naturally depends in particular on the so-called microtiter plate layout on the PCR block.

With a standardized microtiter plate layout, there are 12 x 8 recesses or holes in thermoblocks or the PCR block; these 96-well arrangements are advantageously used in a wide variety of laboratory routines in clinics and basic research. However, thermal blocks with 192, 384 or more cutouts, holes or bores can also be used. With a 96-well layout, for example, samples - according to the standardized labeling of the microtita plate layout (horizontal 1-12, vertical A to H) - at positions A1, B2, C3, A10, B11, C12 and F1, G2, H3 and F10, G11 and H12 are fixed or positioned in such a way that they are operatively connected to these locations on the metal block, with three samples each being attached to the four outer corners in a line. In addition, three samples in particular are positioned at positions D5, D6 and D7. In such a case, it can be provided, for example, that the same number of copies, for example 10,000 copies, is introduced in reaction batches A1, F1, D5, A10 and F10, and in positions B2, B11, G2, G11 and D7 1,000 copies each and 100 copies in the remaining positions.

By disclosing this positioning in a PCR block - designed with a 96-well microtiter plate layout - it is possible for the person skilled in the art to select groupings for all other layouts by routine experimentation or simple consideration.

In a further particularly preferred embodiment of the invention, the arrangement of the PCR reaction mixtures is essentially adapted to the arrangement of the Peltier elements and / or the temperature probes of the thermal cycler.

The PCR reaction batches are advantageously positioned in such a way that they are optimally adapted to the arrangement of the heating and cooling elements, so as to enable the most objective measurement of the annealing and denaturation temperature, the measurement of the individual reaction batches based on the PCR block are arranged, can provide information on all possible reaction sites on the PCR block.

In a further particularly preferred embodiment of the invention, the following reaction is carried out in the PCR reaction mixture,<ul id="ul0001" list-style="dash" compact="compact"><li>Initial denaturation: 1 min, 95 ° C in 20 cycles with the following phases:</li><li>Denaturation: 10 sec, 95 ° C, annealing: 20 sec, 63 ° C, elongation: 20 ° C, 72 ° C and terminal elongation: 5 sec, 72 ° C.</li></ul>

The invention also relates to a referencing kit, which comprises at least one primer with at least one mismatch at the 3 'end and a high-melting PCR template in which the T_M at least one section in the template is greater than 90 ° C. The referencing kit can be used, for example, as a reaction approach for the validation or referencing of PCR blocks. The kit can include information, for example a package insert, which contains information about the use of the individual components of the kit, preferably for the validation and referencing of thermal cyclers.

It is preferred that at least one target DNA is associated with a wall of the reaction mixture, for example a tube. It is particularly preferred that both target DNAs and in particular the respective primers are associated with the wall of the PCR reaction mixture. The kit can comprise, for example, at least one, 10, 20 or more PCR reaction batches in which at least one, preferably both or more, target DNAs are associated on the inner wall.

The invention also relates to the use of the kit or the use of the method according to the invention for carrying out the validation or referencing of thermal cyclers.

The method according to the invention, the kit and the use have several advantages over the prior art:

The method and kit can be used at any time in the laboratory, i.e. under real conditions. This enables regular checking of thermal cyclers as well as immediate use if unsolved problems occur during the PCR.

A particular advantage is the detection of shifts in the temperature control both at temperatures that are too low than at temperatures that are too high or set according to the protocol. Due to the special design of the thermal cycler, temperature inaccuracies and temperature deviations practically always occur in one direction.

With the method presented, a shift towards lower temperatures than set is detected with the reaction for checking the denaturing temperature and a shift towards higher temperatures than set is also detected with the reaction for checking the annealing temperature.

With this system, therefore, displacements in both directions and thus particularly preferably all possible deviations are detected. This method can advantageously be used as a laboratory routine and accordingly as a routine check in a laboratory that requires constant control of its test equipment, such as accredited laboratories or ISO 9001 and others.

In the following, the invention is to be explained at least partially in the figures using an example, without being limited to this example.

Example:

After the PCR has ended, the reaction products are applied to a gel, separated and visualized using a conventional staining reagent. The detection is carried out visually or with a camera (see FIGS. 1 to 4).

In a preferred variant, 5 .mu.l of the reaction are separated by agarose gel electrophoresis and the product is then made visible by staining.

If the annealing temperature was correctly maintained in all block positions, then a clear band can be detected in all reactions with a and b as the starting DNA. The reactions with the starting quantity c only give a weak band.

If the annealing temperature is not observed, more than one band will disappear. This behavior is shown in FIGS. 1 and 2, 63 ° C. being the correct temperature and 66 ° C. being too high an annealing temperature.

If the denaturation temperature was correctly maintained in all block positions, then a clear band can be detected in all reactions with a and b as the starting DNA. The reactions with the starting quantity c only give a weak band.

If the denaturing temperature is not maintained, more than one band disappears (see FIGS. 1 to 4).