Polymerases for nucleotide analogue incorporation
First Claim
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1. A method of sequencing a DNA template, the method comprising:
- a) providing a reaction mixture comprising;
the DNA template,a replication initiating moiety that complexes with or is integral to the template, a recombinant DNA polymerase capable of replicating at least a portion of the template using the moiety in a template-dependent polymerization reaction, wherein the recombinant polymerase comprises an amino acid sequence that is at least 80% identical to SEQ ID NO;
1 and comprises an amino acid substitution at position 375 and an amino acid substitution at position 512, wherein numbering of positions is relative to SEQ ID NO;
1, wherein the recombinant polymerase exhibits polymerase activity, andone or more nucleotide analogues, each of which nucleotide analogues is labeled on a phosphate group, wherein the base moiety of each of the analogues is selected from the group consisting of adenine, thymine, guanine, and cytidine;
b) subjecting the reaction mixture to a polymerization reaction in which the recombinant polymerase replicates at least a portion of the template in a template-dependent manner, whereby the one or more nucleotide analogues are incorporated into the resulting DNA; and
c) identifying a time sequence of incorporation of the one or more nucleotide analogues into the resulting DNA.
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Abstract
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.
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Citations
21 Claims
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1. A method of sequencing a DNA template, the method comprising:
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a) providing a reaction mixture comprising; the DNA template, a replication initiating moiety that complexes with or is integral to the template, a recombinant DNA polymerase capable of replicating at least a portion of the template using the moiety in a template-dependent polymerization reaction, wherein the recombinant polymerase comprises an amino acid sequence that is at least 80% identical to SEQ ID NO;
1 and comprises an amino acid substitution at position 375 and an amino acid substitution at position 512, wherein numbering of positions is relative to SEQ ID NO;
1, wherein the recombinant polymerase exhibits polymerase activity, andone or more nucleotide analogues, each of which nucleotide analogues is labeled on a phosphate group, wherein the base moiety of each of the analogues is selected from the group consisting of adenine, thymine, guanine, and cytidine; b) subjecting the reaction mixture to a polymerization reaction in which the recombinant polymerase replicates at least a portion of the template in a template-dependent manner, whereby the one or more nucleotide analogues are incorporated into the resulting DNA; and c) identifying a time sequence of incorporation of the one or more nucleotide analogues into the resulting DNA. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
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18. A method of making a DNA, the method comprising:
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a) providing a reaction mixture comprising; a template, a replication initiating moiety that complexes with or is integral to the template, a recombinant DNA polymerase capable of replicating at least a portion of the template using the moiety in a template-dependent polymerase reaction, wherein the recombinant polymerase comprises an amino acid sequence that is at least 80% identical to SEQ ID NO;
1 and comprises an amino acid substitution at position 375 and an amino acid substitution at position 512, wherein numbering of positions is relative to SEQ ID NO;
1, wherein the recombinant polymerase exhibits polymerase activity, andat least one nucleotide analogue that is labeled on a phosphate group, wherein the base moiety of the analogue is selected from the group consisting of adenine, thymine, guanine, and cytidine; and b) reacting the mixture such that the polymerase replicates at least a portion of the template in a template-dependent manner, whereby the at least one nucleotide analogue is incorporated into the resulting DNA. - View Dependent Claims (19, 20, 21)
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Specification