Thermal Cycling Profile for Standard PCR
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| Initial denaturation |
| It is very important to denature the template DNA completely. Initial
heating of the PCR mixture for 2 minutes at 94°–95°C is enough to completely
denature complex genomic DNA so that the primers can anneal to the template
as the reaction mix is cooled. If the template DNA is only partially denatured,
it will tend to “snap-back” very quickly, preventing efficient primer annealing
and extension, or leading to “self-priming,” which can lead to false-positive
results. |
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| Denaturation step during cycling |
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Denaturation at 94°–95°C for 20–30 seconds is usually sufficient, but
this must be adapted for the thermal cycler and tubes being used. (For
example, longer times are required for denaturation in 500 µl tubes than
in 200 µl tubes.) If the denaturation temperature is too low, the incompletely
melted DNA “snaps-back” as described earlier, thus giving no access to
the primers. Use a longer denaturation time or higher denaturing temperature
for GC-rich template DNA.
Note: Never use a longer denaturation time than absolutely required for
complete denaturation of template DNA. Unnecessarily long denaturation
times decrease the activity of Taq DNA Polymerase.
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| Primer annealing |
| For most purposes, annealing temperature has to be optimized empirically.
The choice of the primer annealing temperature is probably the most critical
factor in designing a high specificity PCR. If the temperature is too high,
no annealing occurs, but if it is too low, non-specific annealing will increase
dramatically. Primer-dimers will form if the primers have one or more complementary
bases so that base pairing between the 3' ends of the two primers can occur.
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| Primer extension |
| For fragments up to 3 kb, primer extension is normally carried out at
72°C. Taq DNA Polymerase can add approximately 60 bases per second at 72°C.
A 45-second extension is sufficient for fragments up to 1 kb. For extension
of fragments up to 3 kb, allow about 45 seconds per kb. However, these times
may need to be adjusted for specific templates. For improved yield, use
the cycle extension feature of the thermal cycler. For instance, perform
the first 10 cycles at a constant extension time (e.g. 45 s for a 1 kb product).
Then, for the next 20 cycles, increase the extension time by 2–5 s per cycle
(e.g. 50 s for cycle 11, 55 s for cycle 12, etc.). Cycle extension allows
the enzyme more time to do its job, because as PCR progresses, there is
more template to amplify and less enzyme (due to denaturation during the
prolonged high PCR temperatures) to do the extension. |
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| Cycle number |
| In an optimal reaction, less than 10 template molecules can be amplified
in less than 40 cycles to a product that is easily detectable on a gel stained
with ethidium bromide. Most PCRs should, therefore, include only 25 to 35
cycles. As cycle number increases, nonspecific products can accumulate (see
figure below). |
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| Figure 1: Effect of excessive cycling on impure and pure templates
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A PCR product (245 bp amplicon from exon 6 of the dopamine 2 receptor
gene) was reamplified in a series of reactions. In one set of experiments,
the template was not purified before it was used. In the second set, the
template was purified by agarose gel electrophoresis before reamplification.
In both sets, the template was amplified for either 40, 60, or 72 cycles.
Aliquots (8 µl) of the products were analyzed on a 3% agarose gel.
MWM: Molecular Weight Marker; 40, 60, and 72: Number of amplification
cycles.
Result: In both sets, the lowest number of cycles (40) produced the most
specific product. In both the 60 and 72 cycle amplifications, a smear
appeared which contained multimeric “specific” PCR products. Photo courtesy
of U. Finckh and A. Rolfs, Free University of Berlin, Germany
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| Final extension |
| Usually, after the last cycle, the reaction tubes are held at 72°C for
5–15 minutes to promote completion of partial extension products and annealing
of single-stranded complementary products. |