Polymerase enzymes and reagents for enhanced nucleic acid sequencing
AssigneePacific Biosciences of California, Inc.
InventorsDavid R. Rank, Arek Bibillo, Paul Peluso
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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.
Claims25
A composition comprising:
- a modified recombinant Φ29-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 Φ29-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.
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 modified recombinant Φ29-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 Φ29-type DNA polymerase exhibits polymerase activity, and
- one 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.
A method of making a DNA, the method comprising:
- a) providing a reaction mixture comprising:
- a template,
- a replication initiating moiety that complexes with or is integral to the template,
- a modified recombinant Φ29-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 Φ29-type DNA polymerase exhibits polymerase activity, and
- one 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.
Dependencies are inferred from the claim text.