Polymerase enzymes and reagents for enhanced nucleic acid sequencing
First Claim
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1. A composition comprising:
- a modified recombinant Φ
29-type DNA polymerase, which modified recombinant polymerase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO;
1 and comprises an amino acid substitution at position 368 and an amino acid substitution at position 512, wherein numbering of positions is relative to SEQ ID NO;
1, wherein the modified recombinant Φ
29-type DNA polymerase exhibits polymerase activity; and
a 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.
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Abstract
Compositions that include DNA polymerases having increased residence times for nucleotide analogues, particularly modified recombinant Φ29-type DNA polymerases with such increased residence times, are provided. Methods of making the polymerases and of using the polymerases in sequencing and DNA amplification are also provided. Compositions including α-thiophosphate nucleotide analogues with four or more phosphate groups are described, as are methods for determining the sequence of nucleic acid molecules using such analogues.
99 Citations
25 Claims
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1. A composition comprising:
- a modified recombinant Φ
29-type DNA polymerase, which modified recombinant polymerase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO;
1 and comprises an amino acid substitution at position 368 and an amino acid substitution at position 512, wherein numbering of positions is relative to SEQ ID NO;
1, wherein the modified recombinant Φ
29-type DNA polymerase exhibits polymerase activity; anda 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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 25)
- a modified recombinant Φ
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11. 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 modified recombinant Φ
29-type DNA polymerase capable of replicating at least a portion of the template using the moiety in a template-dependent polymerization reaction, wherein the modified recombinant polymerase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO;
1 and comprises an amino acid substitution at position 368 and an amino acid substitution at position 512, wherein numbering of positions is relative to SEQ ID NO;
1, and wherein the modified recombinant Φ
29-type DNA 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 modified 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 (12, 13, 14, 15, 16, 17, 18)
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19. 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 modified recombinant Φ
29-type polymerase capable of replicating at least a portion of the template using the moiety in a template-dependent polymerase reaction, wherein the modified recombinant polymerase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO;
1 and comprises an amino acid substitution at position 368 and an amino acid substitution at position 512, wherein numbering of positions is relative to SEQ ID NO;
1, and wherein the modified recombinant Φ
29-type DNA 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; and b) reacting the mixture such that the polymerase replicates at least portion of the template in a template-dependent manner, whereby the one or more nucleotide analogues are incorporated into the resulting DNA. - View Dependent Claims (20, 21, 22, 23, 24)
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Specification