Method for sequencing using branching fraction of incorporatable nucleotides
First Claim
1. A method of determining which type of nucleotide is incorporated at a site of a template nucleic acid during a template dependent polymerization reaction, the method comprising:
- incorporating a nucleotide into a nucleic acid polymer whereby signal pulses or signatures are generated from branch fraction nonincorporation events from incorporatable nucleotides and, optionally, actual nucleotide incorporation events, for the site;
monitoring a time course of signal pulses or signatures produced by the polymerization reaction; and
,assigning which type of nucleotide is incorporated at the site, using multiple signal pulses or signatures from branch fraction nonincorporation sampling events per nucleotide from incorporatable nucleotides to determine which nucleotide is incorporated at a particular site of the template nucleic acid.
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Accused Products
Abstract
Provided are methods for enhanced sequencing of nucleic acid templates. Also provided are reaction conditions that increase branching fractions during polymerization reactions. Also provided are compositions comprising modified recombinant polymerases that exhibit branching fractions that are higher than the branching fractions of the polymerases from which they were derived. Provided are compositions comprising modified recombinant polymerases that exhibit delayed translocation relative to the polymerases from which they were derived. Also provided are compositions comprising modified recombinant polymerases that exhibit increased nucleotide or nucleotide analog residence time at an active site of the polymerase. Provided are methods for generating polymerases with the aforementioned phenotypes and methods of using such polymerases to sequence a DNA template or make a DNA. Also provided are methods and nucleic acid sequencing systems for determining which labeled nucleotide is incorporated at a site during a template-dependent polymerization reaction.
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Citations
16 Claims
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1. A method of determining which type of nucleotide is incorporated at a site of a template nucleic acid during a template dependent polymerization reaction, the method comprising:
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incorporating a nucleotide into a nucleic acid polymer whereby signal pulses or signatures are generated from branch fraction nonincorporation events from incorporatable nucleotides and, optionally, actual nucleotide incorporation events, for the site; monitoring a time course of signal pulses or signatures produced by the polymerization reaction; and
,assigning which type of nucleotide is incorporated at the site, using multiple signal pulses or signatures from branch fraction nonincorporation sampling events per nucleotide from incorporatable nucleotides to determine which nucleotide is incorporated at a particular site of the template nucleic acid. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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13. A method of determining which of two or more labeled nucleotides is incorporated at a site of a template nucleic acid during a template-dependent polymerization reaction, the method comprising:
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incorporating the nucleotide into a nucleic acid polymer produced by the polymerization reaction, whereby signal pulses or signatures are generated; monitoring the pulses or signatures; and
,using the presence of multiple pulses corresponding to the nucleotide, or identical molecules thereof, to assign which labeled nucleotide is incorporated at the site using mulitple signal pulses or signatures from branch fraction nonincorporation sampling events per nucleotide to determine which nucleotide is incorporated at a particular site of the template nucleic acid. - View Dependent Claims (14)
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15. A method of sequencing a nucleic acid template, the method comprising:
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a) providing a reaction mixture comprising; i) the nucleic acid template; ii) a replication initiating moiety that complexes with or is integral to the template; iii) a modified recombinant nucleic acid polymerase that exhibits an increased branching fraction compared to a corresponding wild-type polymerase, wherein the polymerase is capable of replicating at least a portion of the template using the moiety in a template-dependent polymerization reaction; and iv) one or more nucleotides and/or nucleotide analogs; 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 nucleotides and/or nucleotide analogs are incorporated into the resulting copy nucleic acid; and c) identifying a time sequence of incorporation of the one or more nucleotides and/or nucleotide analogs into the resulting copy nucleic acid using multiple signal pulses or signatures from branch fraction nonincorperation sampling events per nucleotide to determine which nucleotide is incorporated at a particular site of the template nucleic acid.
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16. A method of making a nucleic acid, the method comprising:
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a) providing a reaction mixture comprising; i) a template, ii) a replication initiating moiety that complexes with or is integral to the template, iii) a modified recombinant nucleic acid polymerase that exhibits an increased branching fraction compared to a corresponding wild-type polymerase, wherein the polymerase is capable of replicating at least a portion of the template using the moiety in a template-dependent polyrnerase reaction, and iv) one or more nucleotides and/or nucleotide analogs; and b) reacting the mixture such that the polymerase replicates at least a portion of the template in a template-dependent manner, whereby the one or more nucleotides and/or nucleotide analogs are incorporated into the resulting nucleic acid.
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Specification