Taq and Other Thermostable DNA Polymerases

Abstract

The commercial availability of thermostable DNA dependent DNA polymerase enzymes has been the major factor in facilitating the success of PCR amplification. Initially, the Klenow fragment of DNA-dependent DNA polymerase I (involved in replication and repair in the bacterium Escherichia coli), was employed in PCR amplification [Saiki et al., 1985; Mullis et al., 1992; Saiki et al., 1986]. The Klenow fragment is actually a hydrolytic product of the native E. coli DNA-dependent DNA polymerase enzyme which lacks a 5′-3′ exonuclease activity (Fig. 2.3). The Klenow fragment polymerase was used in the first PCR protocols developed, but has the huge disadvantage of exhibiting an optimum reaction temperature at 37°C and being heat labile at the temperatures used in PCR thermocycling reactions. This lability meant that originally, fresh enzyme had to be added after each and every PCR cycle, making the PCR procedure time consuming, labour intensive and highly prone to contamination. Further, it was impossible to generate PCR fragments longer than 400 bp and further processing (using for example Southern blotting or dot spot hybridization), was usually required to identify the presence/absence of specific amplification products from the mixture of amplimers produced. A breakthrough was achieved however in 1976, when Chien et al. [1976] described a 94kD thermostable DNA-dependant DNA polymerase derived from a eubacterium called Thermus aquaticus, whose natural habitat is hot thermal springs (with ambient temperatures of 70–75°C). This thermostable DNA polymerase or “Taq” enzyme was found to possess similar properties to E. coli DNA dependant DNA polymerase I, with strong homology being found at the amino acid level so: the 3′-OH nucleotide addition site, the dNTP/DNA binding sites, and the 5′-3′ exonuclease sites of the two enzymes (Fig. 7.1). Unfortunately however, though this new Taq polymerase did exhibit some endogenous reverse transcriptase activity, it was found that the 3′-5′ proofreading domain (whereby misincorporated nucleotides are removed from double stranded DNA and replaced by the correct complementary nucleotide) was missing. Moreover, despite the obvious thermoresistant qualities of Taq polymerase, it took a further 2 years for the enzyme to be included in a published PCR protocol [Saiki et al., 1986], after which, there was an explosive increase in interest in PCR mediated amplification protocols. To date, many different types of thermostable DNA dependent DNA polymerases have been discovered in many different thermophilic organisms (Section 7.5), with the majority of these enzymes having their own particular characteristics and being available from commercial suppliers. However, Taq thermostable DNA polymerase (along with its variants) still remains the most widely used polymerase in PCR reactions performed today.