Variants of PCR - DNA Polymerases

DNA Polymerases

There are several DNA polymerases that are used in PCR:

• The Klenow fragment, derived from the original DNA Polymerase I from E. coli, was the first enzyme used in PCR. Because of its lack of stability at high temperature, it needs be replenished during each cycle, and therefore is not commonly used in PCR.

• The bacteriophage T4 DNA polymerase was also initially used in PCR. It has a higher fidelity of replication than the Klenow fragment, but is also destroyed by heat.

• The DNA polymerase from Thermus aquaticus (or Taq), was the first thermostable polymerase used in PCR, and is still the one most commonly used. The enzyme can be isolated from its native source, or from its cloned gene expressed in E. coli.

• The Stoffel fragment is made from a truncated gene for Taq polymerase and expressed in E. coli. It is lacking 5'-3' exonuclease activity, and may be able to amplify longer targets than the native enzyme.

• Faststart polymerase is a variant of Taq polymerase that requires strong heat activation, thereby avoiding non-specific amplification due to polymerase activity at low temperature (see hot-start PCR above).

• Pfu DNA polymerase, isolated from the archean Pyrococcus furiosus, has proofreading activity, and a 5-fold decrease in the error rate of replication compared to Taq. Since errors increase as PCR progresses, Pfu is the preferred polymerase when products are to be individually cloned for sequencing or expression.

• Vent polymerase is an extremely thermostable DNA polymerase isolated from Thermococcus litoralis.

• Tth polymerase is a thermostable polymerase from Thermus thermophilus. It has reverse transcriptase activity in the presence of Mn2+ ions, allowing PCR amplification from RNA targets.