DNA Amplification by the Polymerase Chain Reaction: Molecular Biology Made Accessible - Insect Molecular Genetics - page 308

Taq DNA polymerase is a 94-kDa protein with a temperature optimum of ≈ 75
to 80 °C. It can extend (add on) > 60 nucleotides per second at 70 °C with a
GC-rich 30-mer primer. In a PCR mixture, Taq retains 50% of its activity after ≈ 40
minutes at 94 °C. The use of Taq thus increased the specificity and yield of the
PCR over that possible with the Klenow fragment because the primers could
be annealed and extended at higher temperatures, which eliminated much of
the nonspecific amplification. Longer PCR products were produced because the
secondary structure of the template DNA was eliminated at these higher tem-
peratures, as well. Fragments of ≈ 500bp can be synthesized with the Klenow
fragment, but fragments up to 10 kb sometimes can be produced with Taq .
8.3.5 Other Thermostable DNA Polymerases
Genetically engineered variants of Taq have been developed and DNA polymer-
ases from other sources now are available commercially in native and cloned
form ( Engelke et al. 1990, Erlich et al. 1991, Perler et al. 1996 ). For example, a
recombinant T. aquaticus polymerase called the “Stoffel fragment” persists at
97.5 °C and exhibits optimal activity over a broad range of Mg 2 + concentrations.
Other thermostable DNA polymerases have been isolated from Eubacteria
( Thermus and Bacillus ) and Archaea ( Thermococcus , Pyrococcus , and Sulfolobus ).
Many are commercially available and have specific attributes such as 3 ′ to 5 ′
exonuclease activity, “proofreading” ability, different molecular weights, dif-
ferent stabilities and temperature optima ( Perler et al. 1996 ). For example, a
thermostable enzyme isolated from T. thermophilus can reverse-transcribe RNA
efficiently at high temperatures. The thermostability of this enzyme appears to
minimize the importance of secondary structure in the RNA template and allow
efficient cDNA synthesis at high temperatures.
A DNA polymerase isolated from the archaebacterium S. acidocaldarius car-
ries out polymerization at 100 °C, which facilitates amplification of DNA
regions with secondary structure ( Arnheim and Erlich 1992 ). Polymerases from
Thermoplasma acidophilum , Thermococcus litoralis , and Methanobacterium
thermoautotrophicum have 3 ′ to 5 ′ exonuclease activities, which means that
they can proofread, reducing the rate of misincorporation or errors.
8.3.6 Primers are Primary
Although all the components of a PCR are important, primers are truly crucial
( Table 8.2 ). Well-designed primers can result in 100- to 1000-fold increases in
sensitivity ( He et al. 1994a,b ). As a rule, longer primers are better than shorter
for increasing specificity, but length is not the only consideration (Yuryev 2007).