Polymerase chain reaction fidelity

Ling, L.L., P. Keohavong, C. Dias, and W.G. Thilly, Optimization of the polymerase chain reaction with regard to fidelity modified T7,... 

Evolution has been shown to be an efficient tool for the improvement of enzymes and pathways. Stochastic approaches to introduce point mutations in genes include error prone polymerase chain reaction (ePCR),11 the use of low-fidelity (mutator) strains,12 and chemical mutagens.13... 

The DNA polymerases of T. littoralis and P. furiosus have been marketed for use in DNA amplification by the polymerase chain reaction (PCR) method as the Vent and pfu DNA polymerases, respectively. These enzymes are more accurate in vitro than the Thermus aquaticus (Taq) DNA polymerase, both in classical fidelity tests [132] and in PCR [133,134]. Indeed, they have an associated 3 to 5 exonuclease activity involved in proof-reading, whereas the Taq polymerase is devoid of such activity. 

Eckert KA, Kunkel TA (1991). DNA pol3unerase fidelity and the polymerase chain reaction. PCR Methods AppL, 1 17-24. 

High-fidelity polymerase chain reaction (PCR) amplification kit (e.g., Phusion polymerase (New England BioLabs , Inc. [NEB])). 

Step 3 - Primer extension The synthesis of DNA is catalysed by the polymerase. The 3 -end of the hybridised primer is extended along the original DNA strand by continuous incorporation of the complementary nucleotides into the chain. Thus, a complementary DNA strand is formed. Primer extension proceeds from the 3 -end to the 5 -end. This extension reaction stops as soon as the complementary strand has been completed. It has been empirically shown that the fidelity of the construction of the complementary strand improves at 72 °C. Hence, the temperature is usually adjusted to this value for the primer extension step. 

Role of RNA intermediates (Herbert and Rich, 1999). The mutation rate of a genome is likely to increase when genetic information is passed through RNA whether RNA is a viral genome or a retrotransposon because RNA polymerase reaction is neither edited nor subject to post-replicative repair. In addition, hotspots of a genetic chain in RNA retrotransposons can result from nomandom patterns of a decreased fidelity strand transfer to other templates and untemplated extensions. 

The lag time depends explicitly on the concentration of the next correct dNTP, giving rise to a next nucleotide effect (10,11). High concentrations of dNTPs (>200 fiM) increase the error rate by driving the polymerase reaction (12) a decrease in lag time reduces the chance of error discrimination by the polymerase at each extension step and consequently the chance of mismatching nucleotide excision by the exonuclease. Polymerase fidelity tends to increase at lower pH (e.g., pH 6.5 versus pH 8.5) as a result of decreased efficiency of chain elongation. Note that a marked increase of error rate is also observed when the concentrations... 

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