Pacific Biosciences of California, Inc.

Harold Ray Lee

Keith P Bjornson

Molly Min He

Robin A Emig

Fred C Christians

Lei Jia

Insil Park

Jonas Korlach

Arek Bibillo

Paul S Peluso

Sonya Clark

Stephen Wilson Turner

Erik Miller

Satwik Kamtekar

Jeremiah Hanes

John Lyle

Andrei Fedorov

John M Lyle

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

The Application relates to methods, and systems for nucleotide sequencing comprising producing polymerase reactions that comprise both catalytic and non-catalytic divalent metal ions. Polymerase/template/primer complexes are immobilized on a substrate. The complexes are exposed to a solution comprising a non-catalytic metal and nucleotides labeled with a detectable label on a portion of the nucleotide that is released upon incorporation. The cognate nucleotide is sequestered in the active site of the polymerase, unable to proceed to incorporation. After observing the sequestered cognate nucleotide, the complex is exposed to a catalytic metal, resulting in the incorporation of the bound cognate nucleotide and consequent release of the label resulting in a single-base extended primer.

Step-wise nucleic acid sequencing with catalytic and non-catalytic metals

The Application relates to compositions, kits, methods, and systems for nucleotide sequencing comprising producing polymerase reactions that comprise both catalytic and non-catalytic divalent metal ions. Effective ratios and amounts of catalytic and non-catalytic divalent metal ions are described.

Nucleic acid synthesis compositions and methods and systems for using same

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.

Compositions, kits, methods and systems for nucleotide sequencing comprising producing polymerase reactions that exhibit two kinetically observable steps within an observable phase of the polymerase reaction. Two slow step systems can be produced, for example, by selecting the appropriate polymerase enzyme, polymerase reaction conditions including cofactors, and polymerase reaction substrates including the primed template and nucleotides.

Two slow-step polymerase enzyme systems and methods

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Sunnyvale, CA, United States of America

Harold Ray Lee