Polymerase idling method for single molecule DNA sequencing
AssigneeAgilent Technologies, Inc.
InventorsBrian Jon Peter, Joel Myerson
Life of this patent
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Abstract
A method for sequencing a nucleic acid is provided. In certain embodiments the method comprises obtaining a duplex comprising a nucleic acid and a primer, wherein the primer has a nuclease resistant 3′ end, combining the duplex with a chain terminator nucleotide and a proof-reading polymerase to produce a reaction in which the polymerase idles on the added chain terminator nucleotide, identifying the chain terminator nucleotide added to the end of the primer; and adding a nuclease-resistant nucleotide to the end of the primer after the polymerase has idled on and removed the added chain terminator nucleotide, thereby producing a duplex comprising the template and an extended primer that has a nuclease resistant 3′ end.
Claims20
A method of sequencing DNA, comprising:
- (a) obtaining a duplex comprising a nucleic acid and a primer;
- (b) combining the duplex with one or more nucleoside polyphosphates, one or more chain terminator nucleotides, a DNA polymerase, and inorganic pyrophosphate to produce a reaction in which the polymerase repeatedly adds a chain terminator nucleotide onto the end of the primer using the nucleic acid as a template, and then removes the chain terminator nucleotide by pyrophosphorolysis;
- and
- (c) identifying the chain terminator nucleotide added to the end of the primer;
- wherein:
- i) the probability of incorporating a chain terminator nucleotide is greater than the probability of incorporating a nucleoside polyphosphate.
Dependencies are inferred from the claim text.