Optimization Of Pcr Conditions For Long Pcr

Abstract

PCR has proven to be one of the most powerful tools in biotechnology field. However, there have been difficulties that challenged the traditional PCR. Target length and fidelity limitations have been encountered. Optimizing PCR additives have shown improvements in PCR results. PCR polymerases is another approach. Traditional Taq has length limitation. Polymerases with 3’-exonuclease activity have high fidelity performance while compromising on processivity. The combination of Taq polymerase with a significant proportion to a 3’-exonuclease polymerase have exhibited length improvement of PCR products. In this experiment, glycerol has shown to be the most effective additive when using concentration of between 6% - 8% in reducing smear and enhancing results of long PCR. Modified Taq polymerase with a 3’-exonuclease domain (FU14) has proven to be a potent polymerase in amplifying long DNA fragment. In this experiment FU14 have exceed its limitation and amplified successfully up to 9kb. Although the combination of Taq and FU14 did not performed as expected, it has shown some potential in improving band thickness of long PCR.