Biology Reference
In-Depth Information
8.3.9 PCR Automation
The PCR involves repeated cycles with at least two, and generally three, tempera-
tures ( Figure 8.2 ). A high temperature is needed to denature (separate the two
strands) the DNA template and subsequent product molecules. The lower temper-
ature should allow annealing of the primer to the denatured ss DNA template. A
third intermediate temperature close to the optima for DNA polymerase function
is used for the extension (synthesis of the complementary sequence) phase. The
annealing temperatures should be neither too low nor too high. An algorithm can
be used to determine the optimal annealing temperature for a given pair of prim-
ers and template DNA based on the GC content of the primer-template sequences
( Rychlik et al. 1990 ). This algorithm is available in computer programs ( Osborne
1992 ) and at many websites of companies that provide primer synthesis services.
A variety of commercial thermal cyclers controlled by microprocessors auto-
mate the rapid and precise heating and cooling required for maximum effi-
ciency of the PCR. Three basic categories of commercial temperature cyclers are
available in which the reaction is 1) heated and cooled by fluids, 2) heated by
electric resistances and cooled by fluids, or 3) heated by electric resistances and
cooled by semiconductors. Accuracy and reproducibility in temperature control
should be a concern when choosing a temperature cycler. Temperature cyclers
are designed for use with 0.5- and 0.2-ml microfuge tubes or with 96-well plates.
Maintaining a close fit between the walls of the block and the microfuge tube
or well plate is critical in maintaining accurate temperatures. Filling the wells with
glycerol or mineral oil will encourage the transfer of heat. Thermocyclers should
be checked periodically to determine their accuracy and calibrated if needed; dif-
ferences of even 1-2 °C can be significant.
Commercially available temperature cyclers cost US$3000-$8000. They differ
in the design of the cooling system, control of ramping time between temper-
ature steps, memory capacity for program storage, sequential linking of pro-
grams, and capacity of the heating block to hold different numbers of samples
for amplification. Machines that do not provide a uniform temperature across a
heating block can lead to variation in outcomes from the reactions taking place
in different samples. Different models or brands of temperature cyclers, while
ostensibly programmed to produce the same temperature profiles, may not be
equivalent which can alter the outcomes of the PCR.
8.3.10 Specificity of the PCR
The specificity (or fidelity) of the PCR based on DNA synthesis by the Klenow
DNA polymerase is low. The use of Taq and other DNA polymerases not only
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