Generation of modified polymerases for improved accuracy in single molecule sequencing

US8420366B2 · kind B2 · utility

Assignee

• PACIFIC BIOSCIENCES OF CALIFORNIA, INC. · US

Inventors

• Sonya Clark · Oakland, US
• Arek Bibillo · Cupertino, US
• Paul Peluso · East Palo Alto, US
• Fred Christians · Los Altos Hills, US
• Molly He · San Diego, US
• Insil Park · Fremont, US
• Harold Lee · Holly Springs, US
• Keith Bjornson · Fremont, US
• Lei Jia · Palo Alto, US
• Robin Emig · Belmont, US

Key dates

• Filing date: Sep 30, 2010
• Grant date: Apr 16, 2013
• Expiry date: Feb 22, 2031

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12Q2521/101
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

Provided are compositions comprising recombinant DNA polymerases that include amino acid substitutions, insertions, deletions and/or heterologous or exogenous features that confer modified properties upon the polymerase for enhanced single molecule sequencing. Such properties can include reduced reaction rates at one or more steps of the polymerase kinetic cycle, increased closed polymerase/DNA complex stability, enhanced metal ion coordination, reduced exonuclease activity, decreased branching fractions, and the like. Polymerases that exhibit branching fractions that are less than the branching fractions of the polymerases from which they were derived, or branching fractions that are less than about 25% for a phosphate-labeled nucleotide analog, are also provided. Also provided are nucleic acids which encode the polymerases with the aforementioned phenotypes, as well as methods of using such polymerases to make a DNA or to sequence a DNA template.