Polymerase compositions and methods

A method for detecting one or more nucleotide incorporations onto the end of a single nucleic acid molecule, comprising:

• contacting a target nucleic acid molecule with a modified polymerase comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:
• 7 with one or more labeled nucleotides under conditions where at least one labeled nucleotide is incorporated by the polymerase onto the end of an extending nucleic acid molecule, wherein the label of at least one labeled nucleotide emits a signal indicative of nucleotide incorporation;
• and
• detecting the one or more signals indicative of nucleotide incorporation,
• wherein the modified polymerase has amino acid leucine at position 383 and amino acid asparagine at position 384 and one or more additional amino acid modifications, and the modified polymerase exhibits increased polymerase activity relative to the unmodified reference B103 polymerase, SEQ ID NO:
• 6, and wherein the one or more additional amino acid modifications of the modified polymerase consist of:
• i) one or more amino acid substitutions, additions or deletions at one or more positions selected from the group consisting of:
• 9, 11, 12, 58, 59, 63, 162, 166, 377 and 385 and the modified polymerase exhibits reduced exonuclease activity relative to an unmodified B103 polymerase, SEQ ID NO:
• 6;
• ii) one or more amino acid substitutions at one or more positions selected from the group consisting of:
• 365, 370, 371, 372, 481, 507, and 509, wherein the one or more amino acid substitutions at position 372 is selected from the group consisting of K372G, K372T, K372S, K372R, K372A, K372Q, K372W, K372Y, and K372F, and the modified polymerase exhibits an increased branching ratio and/or increased nucleotide binding affinity relative to a reference modified polymerase of SEQ ID NO:
• 7;
• and/or
• iii) an amino acid substitution at position 380 and the modified polymerase exhibits increased tpo1 value (or decreased kpo1 value) relative to the unmodified B103 polymerase, SEQ ID NO:
• 6.

A method for sequencing a nucleic acid molecule, comprising:

• (a) conducting a polymerase reaction comprising contacting a target nucleic acid molecule with a modified polymerase comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:
• 7 with one or more labeled nucleotides under conditions where at least one labeled nucleotide is incorporated by the polymerase onto the end of an extending nucleic acid molecule, wherein the target nucleic acid molecule is paired with a polymerization initiation site and wherein the label of at least one labeled nucleotide emits a signal indicative of nucleotide incorporation;
• and
• (b) detecting a time sequence of nucleotide incorporations from the one or more signals indicative of nucleotide incorporation;
• and
• (c) determining the identity of one or more incorporated nucleotides, thereby determining some or all of the nucleotide sequence of a single nucleic acid molecule,
• wherein the modified polymerase has amino acid leucine at position 383 and amino acid asparagine at position 384 and one or more additional amino acid modifications, and the modified polymerase exhibits increased polymerase activity relative to the unmodified reference B103 polymerase, SEQ ID NO:
• 6, and wherein the one or more additional amino acid modifications of the modified polymerase consist of:
• i) one or more amino acid substitutions, additions or deletions at one or more positions selected from the group consisting of:
• 2-9, 11, 12, 58, 59, 63, 162, 166, 377 and 385 and the modified polymerase exhibits reduced exonuclease activity relative to an unmodified B103 polymerase, SEQ ID NO:
• 6;
• ii) one or more amino acid substitutions at one or more positions selected from the group consisting of:
• 365, 370, 371, 372, 481, 507, and 509, wherein the one or more amino acid substitutions at position 372 is selected from the group consisting of K372G, K372T, K372S, K372R, K372A, K372Q, K372W, K372Y, and K372F, and the modified polymerase exhibits an increased branching ratio and/or increased nucleotide binding affinity relative to a reference modified polymerase of SEQ ID NO:
• 7;
• and/or
• iii) an amino acid substitution at position 380 and the modified polymerase exhibits increased tpo1 value (or decreased kpo1 value) relative to the unmodified B103 polymerase, SEQ ID NO:
• 6.