DNA Amplification by the Polymerase Chain Reaction: Molecular Biology Made Accessible - Insect Molecular Genetics - page 318

simplifies the PCR but also increases the specificity and overall yield. The higher
temperature optimum for Taq ( ≈ 75 °C) allows the use of higher temperatures
for primer annealing and extension, which increases the stringency of the reac-
tion and minimizes the extension of primers that are mismatched with the tem-
plate DNA.
The increase in specificity with the use of Taq also results in an increased
yield of the target fragment because competition by nontarget products for
DNA polymerase and primers is reduced. In the later cycles, the amount of poly-
merase may no longer be sufficient to extend all the annealed primer-template
complexes in a single cycle interval, which results in reduced efficiency and a
“plateau.” This plateau is reached after ≈ 30 cycles when Taq is used rather than
after 20 when the Klenow fragment is used.
Modifications of the standard PCR can enhance the outcome. Adjusting the
annealing temperature can control the stringency of the annealing step; high-
temperature annealing and extension ( > 55 °C) and a balanced ratio of Mg 2 + and
dNTP concentrations give the greatest fidelity in the final product ( Table 8.2 ).
Various additives such as DMSO (2-5%), PEG 6000 (5-15% polyethylene glycol),
glycerol (5-20%), nonionic detergents and formamide (5%) can be incorporated
into the reaction to increase specificity ( Roux 1995 ).
Optimizing the annealing temperature and minimizing the incubation time
during the annealing and extension steps limits the amount of mispriming.
Reducing primer and Taq concentrations also reduces mispriming. Changing
the MgCl 2 concentration can increase specificity by allowing a higher annealing
temperature, which increases the stringency of the reaction.
Although Taq has no 3 ′ to 5 ′ exonuclease (proofreading) activity, its error
rate is low compared with that of the Klenow fragment ( Mullis et al. 1986,
Keohavong and Thilly 1989 ), because Taq has a 5 ′ to 3 ′ exonuclease activity dur-
ing polymerization ( Erlich et al. 1991 ). Current estimates of misincorporation
rates are 10 − 5 nucleotides per cycle under optimized conditions. Taq appears to
extend a mismatched primer-template significantly less efficiently than a cor-
rect primer-template. Misincorporated bases cannot be removed and this can
promote termination of the extending DNA chain, which prevents propagation
of the errors in subsequent PCR cycles but lowers the yield of the PCR. Because
the accumulation of mutations in the PCR product is proportional to the num-
ber of DNA replications, the fewer cycles that are required to provide an ade-
quate yield of DNA the better. Starting with adequate amounts of template (but
not too much) reduces the number of PCR cycles required to produce a specific
amount of product, and hence the amount of misincorporations.