Abstract: A purified recombinant thermostable DNA polymerase polymerase which exhibits at least about 80% activity at salt concentations of 50 mM and greater, at least about 70% activity at salt concentrations of 25 mM and greater, and having a processivity of about 30 nucleotides per binding event. An isolated nucleic acid that encodes the thermostable DNA polymerase, as well as a recombinant DNA vector comprising the nucleic acid and a recombinant host cell transformed with the vector, are also disclosed. A method of sequencing DNA using the DNA polymerase as well as a kit for sequencing DNA is also disclosed.

Abstract: Polymerases from the Pol I family which are able to efficiently use ddNTPs have demonstrated a much improved performance when used to sequence DNA. A number of mutations have been made to the gene coding for the Pol II family DNA polymerase from the archaeon Pyrococcus furiosus with the aim of improving ddNTP utilisation. “Rational” alterations to amino acids likely to be near the dNTP binding site (based on sequence homologies and structural information) did not yield the desired level of selectivity for ddNTPs. However, alteration at four positions (Q472, A486, L490 and Y497) gave rise to variants which incorporated ddNTPs better than the wild type, allowing sequencing reactions to be carried out at lowered ddNTP:dNTP ratios. Wild type Pfu-Pol required a ddNTP:dNTP ratio of 30:1; values of 5:1 (Q472H), 1:3 (L490Y), 1:5 (A486Y) and 5:1 (Y497A) were found with the four mutants; A486Y representing a 150-fold improvement over the wild type.