Biology Reference
In-Depth Information
8.3.2 PCR Power
The power of the PCR is based on the fact that the products of one replication
cycle serve as a template for the next. Each successive cycle, in theory, doubles
the number of DNA molecules synthesized in the previous cycle, resulting in
the exponential accumulation of the target DNA at approximately 2 n , where
n is the number of cycles. In practice, the PCR is never 100% efficient and less
product will be produced. The early cycles are less efficient than the middle
cycles because precisely defined template strands occur only after the first few
cycles ( Figure 8.1B ). Late in the PCR, the reduced availability of reaction com-
ponents may limit the yield. Reaction components that are reduced include
primer concentration, dNTPs, or DNA polymerase ( Czerny 1996 ). Primers and
DNA polymerase can be degraded by the multiple heating cycles and dNTPs can
be used up or degraded.
8.3.3 Standard PCR Protocols
Table 8.1 gives a procedure suitable for amplifying genomic DNA from
Drosophila and demonstrates the relative simplicity of the technique. Table 8.2
discusses some of the issues that must be considered in setting up new PCRs.
The PCR is performed using commercially available temperature cyclers that
allow the programming of the three fundamental reaction temperatures dur-
ing denaturing , annealing , and extension ( Figure 8.2 ). A typical amplification
cycle involves denaturing the template DNA at 94 °C for 20 seconds, annealing
the primers to the template at 55 °C for 20 seconds, and extending (or synthe-
sizing) the DNA at 72 °C for 30 seconds. Because the instruments require time
to heat and cool to a specific temperature, each actual cycle time may require
10 minutes or more, depending upon the specific machine used. If 25 cycles are
performed, the total time will be 4 hours and the target DNA will have been
amplified 1 million-fold, assuming a doubling has occurred in each cycle.
8.3.4 DNA Polymerases
The PCR, as first described in 1985, used the Klenow fragment of Escherichia coli
DNA polymerase I to produce copies of target DNA ( Saiki et al. 1985, Mullis and
Faloona 1987 ). Because the Klenow fragment is heat sensitive, fresh enzyme had
to be added to each cycle, making the PCR an exceedingly tedious procedure!
The efficiency and fidelity of the PCR was dramatically improved by using a
heat-resistant polymerase ( Taq DNA polymerase ) so that the procedure could
be carried out at high temperatures without having to add new enzyme before
Search WWH ::




Custom Search