Dideoxynucleotide-triphosphate utilization by the hyper-thermophilic DNA polymerase from the archaeon Pyrococcus furiosus

US6333183A · kind A · utility

Assignee

• Amersham Pharmacia Biotech, Inc. · SE

Inventors

• Steven L. Evans · Pocahontas, US
• Joseph Mamone · Fryeburg, US
• Maria Davis · Princeton, US
• Bernard Connolly · Lymington, GB

Key dates

• Filing date: Nov 17, 2000
• Grant date: Dec 25, 2001
• Expiry date: Nov 17, 2020

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12N9/1252
• WIPO field: Biotechnology
• WIPO sector: Chemistry

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. A486, L490 and Y407 are on an .alpha.-helix that lines the dNTP binding groove, but the side chains of the three amino acids point away from this groove; Q472 is in a loop that connects this .alpha.-helix to a second long helix. None of the four a…