Pacific Biosciences of California, Inc.

Insil Park

Fred C Christians

Sonya Clark

Paul S Peluso

Arek Bibillo

John Lyle

Molly Min He

Erik Miller

Satwik Kamtekar

Robin A Emig

Thang Pham

Lei Jia

Geoff Otto

David R Rank

David Kagen Hanzel

Keith P Bjornson

Walter H Lee

Harold Ray Lee

Arekadiusz Bibillo

Lei Sun

Paul Mitsis

Stephen Wilson Turner

Jonas Korlach

Jeremiah Hanes

Andrei Fedorov

Devon Murphy

John M Lyle

Fred Christians

Paul Peluso

Harold Lee

Keith Bjornson

Robin Emig

Molly He

David K Hanzel

Provided are compositions comprising recombinant DNA polymerases that include amino acid substitutions, insertions, deletions, and/or exogenous features that confer modified properties upon the polymerase for enhanced single molecule sequencing. Such properties can include enhanced metal ion coordination, reduced exonuclease activity, reduced reaction rates at one or more steps of the polymerase kinetic cycle, decreased branching fraction, altered cofactor selectivity, increased yield, increased thermostability, increased accuracy, increased speed, increased readlength, and the like. 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.

Recombinant polymerases for improved single molecule sequencing

Compositions that include polymerases with features for improving entry of nucleotide analogues into active site regions and for coordinating with the nucleotide analogues in the active site region are provided. Methods of making the polymerases and of using the polymerases in sequencing and DNA replication and amplification as well as kinetic models of polymerase activity and computer-implemented methods of using the models are also provided.

Polymerases for nucleotide analogue incorporation

Provided are compositions comprising recombinant DNA polymerases that include amino acid substitutions, insertions, deletions, and/or exogenous features that confer modified properties upon the polymerase for enhanced single molecule sequencing. Such properties include increased resistance to photodamage, and can also include enhanced metal ion coordination, reduced exonuclease activity, reduced reaction rates at one or more steps of the polymerase kinetic cycle, decreased branching fraction, altered cofactor selectivity, increased yield, increased thermostability, increased accuracy, increased speed, increased readlength, and the like. 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.

Recombinant polymerases with increased phototolerance

Methods, Compositions, and Systems are provided for obtaining polymerase-template complex mixtures with improved levels of active polymerase. In some aspects active polymerase-template complex is separated by affinity for nucleoside phosphate moieties attached to a resin. In some aspects, a polymerase-template complex is exposed to reaction conditions in which a complementary strand to the template is produced. The extended reaction mixture is purified by reaction with a resin comprising nucleoside phosphate moieties. This purified mixture can be loaded onto substrates and can be used for further analyzes including single molecule sequencing.

Purified polymerase / template complexes

Compositions that include polymerases with features for improving entry of nucleotide analogs into active site regions and for coordinating with the nucleotide analogs in the active site region are provided. Methods of making the polymerases and of using the polymerases in sequencing and DNA replication and amplification as well as kinetic models of polymerase activity and computer-implemented methods of using the models are also provided.

Provided are compositions comprising modified DNA polymerases that exhibit improved photostability compared to the parental polymerases from which they were derived. Provided are methods for generating enzymes, such as DNA polymerases, with the aforementioned phenotype. Provided are methods of using polymerases with increased resistance to photodamage to make a DNA or to sequence a DNA template.

Enzymes resistant to photodamage

Methods, Compositions, and Systems are provided for obtaining polymerase-template complex mixtures with improved levels of active polymerase. In some aspects, methods are described in which a polymerase-template complex is exposed to reaction conditions in which a complementary strand to the template is produced. The extended reaction mixture is purified, for example by gel filtration chromatography to produce a mixture of polymerase-template complex having a higher active fraction. This purified mixture can be used for further analyses including single molecule sequencing.

Purified extended polymerase/template complex for sequencing

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.

Generation of modified polymerases for improved accuracy in single molecule sequencing

Provided are compositions comprising modified recombinant 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. Also provided are compositions comprising modified recombinant polymerases that exhibit closed polymerase/DNA complexes with increased stability relative to the parental polymerases. Also provided are compositions comprising modified recombinant polymerases that exhibit decreased rate constants relative to the parental polymerases. Provided are methods for generating polymerases with the aforementioned phenotypes. Provided are methods of using such polymerases to make a DNA or to sequence a DNA template.

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Fremont, CA, United States of America

Insil Park