Investigating thermostable DNA polymerases for PCR-based applications

Abstract

Thermostable DNA polymerases are essential components of the polymerase chain reaction (PCR), a technique widely applied across the entire biosciences. The work presented in this thesis improves understanding of the function and properties of these enzymes with the aim of developing improved formulations for biotechnological applications. The accuracy with which polymerases replicate DNA is essential for their application in PCR. A plasmid-based DNA polymerase fidelity assay, based on a gapped plasmid template containing the lacZα gene, has been developed. This technique, a marked improvement on previous methods, enables straightforward determination of any polymerase’s fidelity. The functions of two loops in archaeal family-B DNA polymerases, located in the thumb domain responsible for double-stranded DNA binding, have been elucidated, revealing a role in the control of polymerase and proof-reading exonuclease activities. Site-directed mutagenesis, combined with kinetic and binding experiments, was used for this purpose. The family-B DNA polymerase from the archaeon Pyrococcus furiosus has low processivity, limiting its ability to amplify long stretches of DNA. The processivity of this enzyme was increased by changing a number of amino acids to those observed in the more processive polymerase from Thermococcus kodakarensis. Several mutants have been identified with increased processivity and improved performance in PCR. Reverse transcription PCR (RT-PCR) typically requires the use of a mesophilic reverse transcriptase to generate cDNA from RNA, which is then amplified by a thermostable DNA polymerase in PCR. Through the use of compartmentalised self-replication (CSR) and rational design, generation of a DNA polymerase with reverse transcriptase activity capable of single tube RT-PCR was attempted.