Generation of modified polymerases for improved accuracy in single molecule sequencing
First Claim
1. A composition comprising a recombinant DNA polymerase, which recombinant DNA polymerase comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:
- 1, which recombinant DNA polymerase comprises a glutamic acid residue, an aspartic acid residue, a histidine residue, a lysine residue, an arginine residue, a glutamine residue, or a tyrosine residue at position A484, wherein identification of positions is relative to SEQ ID NO;
1, and which recombinant DNA polymerase exhibits polymerase activity.
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Accused Products
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.
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Citations
30 Claims
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1. A composition comprising a recombinant DNA polymerase, which recombinant DNA polymerase comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:
- 1, which recombinant DNA polymerase comprises a glutamic acid residue, an aspartic acid residue, a histidine residue, a lysine residue, an arginine residue, a glutamine residue, or a tyrosine residue at position A484, wherein identification of positions is relative to SEQ ID NO;
1, and which recombinant DNA polymerase exhibits polymerase activity. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 30)
- 1, which recombinant DNA polymerase comprises a glutamic acid residue, an aspartic acid residue, a histidine residue, a lysine residue, an arginine residue, a glutamine residue, or a tyrosine residue at position A484, wherein identification of positions is relative to SEQ ID NO;
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28. A composition comprising a recombinant DNA polymerase, which recombinant polymerase comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:
- 1;
which recombinant polymerase comprises an alanine residue at position 253, a tyrosine residue at position 375, a tyrosine residue at position 512, and a glutamic acid residue at position 484, wherein numbering of positions is relative to SEQ ID NO;
1; and
which recombinant polymerase exhibits polymerase activity.
- 1;
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29. A composition comprising a recombinant DNA polymerase, which recombinant polymerase comprises the amino acid sequence of SEQ ID NO:
- 82.
Specification