Improving dideoxynucleotide-triphosphate utilisation by the hyper-thermophilic DNA polymerase from the archaeon Pyrococcus furiosus.

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

SUBMITTER: Evans SJ 

PROVIDER: S-EPMC102620 | biostudies-literature | 2000 Mar

REPOSITORIES: biostudies-literature