Polymerase chain reaction (PCR) is probably the most popular application in contemporary molecular biology and diagnostics. The key components of PCR are thermostable DNA polymerases, which synthesize new DNA complementary to a DNA matrix. There are many different polymerases, which are used in PCR. Even though Taq DNA polymerase was the first enzyme employed in PCR and many new enzymes were discovered since that time, this polymerase continues to be the most popular and widely used in majority of PCR applications due to its robustness and efficiency as well as easy and cost efficient production process. Taq DNA polymerase has been studied very intensively and there is a lot of biochemical as well as structural data available on it. Within the course of these studies many different mutations of Taq DNA polymerase have been created and studied, which in one or another way improve properties of this enzyme. Some mutations are important for enzyme fidelity (U.S. Pat. Nos. 6,395,524, 6,602,695 and 5,614,365), some alter 5′-3′ exonuclease activity (U.S. Pat. No. 5,466,591), change enzyme properties related to labeled nucleotide incorporation (Brandis et al., 1998), make the enzyme “cold sensitive” (Barnes and Kermekchiev, 2000) or increase polymerase resistance to different PCR inhibitors (Kermekchiev and Barnes, 2004; Kermekchiev and Kirilova, 2006). Such mutants of Taq DNA polymerase are useful in qPCR, DNA sequencing, amplification of DNA samples containing various PCR inhibitors (dye, blood, soil). For example, SYBR Green I intercalating dye is used in qPCR. This inhibits Taq DNA polymerase and can decrease PCR efficiency and sensitivity. Increased polymerase resistance to SYBR Green I may be associated with increased enzyme resistance to other PCR inhibitors from blood and soil (Kermekchiev et al, 2009; Zhang et al, 2010).
Mutation at various different amino acid positions in the Taq DNA polymerase are known to improve various different properties. These include K219, K225, E520, D578, A608 (Brandis et al., 1998; Holliger et al., 2001), S515 (Hardin et al., 2006), A521, V529, Q592 (Brandis et al., 1998) and S543 (Jestin et al., 2005; Vatta et al., 2005). In one example, the positively charged Taq DNA polymerase mutation E507K is known to improve the RNA target dependent activity by 50% compared to the parent enzyme.
Currently PCR represents one of the fastest growing segments of molecular biology applications market. New applications for PCR and new variants of PCR are being developed and introduced for research and diagnostic applications, such as fast qPCR, digital PCR and direct sample-to-PCR which require novel enzymatic properties. Therefore, there is a need in the industry for Taq DNA polymerase derivatives possessing novel, improved properties.
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