Short cycle methods for sequencing polynucleotides
DC CAFCFirst Claim
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1. A method for sequencing a nucleic acid template, the method comprising the steps of:
- (a) exposing a nucleic acid template hybridized to a primer to (i) a polymerase capable of catalyzing nucleotide addition to said primer, and (ii) a labeled nucleotide that is not a chain terminating nucleotide, under reaction conditions and for a time such that on average only one nucleotide is added to said primer by said polymerase to produce an extended primer incorporating said labeled nucleotide;
(b) identifying said single labeled nucleotide incorporated in step (a);
(c) neutralizing label in said single labeled nucleotide incorporated in step (a);
(d) repeating steps (a), (b) and (c) at least once; and
(e) determining a sequence of said template based upon the order of incorporation of said labeled nucleotides.
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Abstract
The invention provides methods for sequencing a polynucleotide comprising stopping an extension cycle in a sequence by synthesis reaction before the reaction has run to near or full completion.
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Citations
12 Claims
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1. A method for sequencing a nucleic acid template, the method comprising the steps of:
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(a) exposing a nucleic acid template hybridized to a primer to (i) a polymerase capable of catalyzing nucleotide addition to said primer, and (ii) a labeled nucleotide that is not a chain terminating nucleotide, under reaction conditions and for a time such that on average only one nucleotide is added to said primer by said polymerase to produce an extended primer incorporating said labeled nucleotide; (b) identifying said single labeled nucleotide incorporated in step (a); (c) neutralizing label in said single labeled nucleotide incorporated in step (a); (d) repeating steps (a), (b) and (c) at least once; and (e) determining a sequence of said template based upon the order of incorporation of said labeled nucleotides. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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12. A method for simultaneously sequencing plural nucleic acid templates, the method comprising the steps of:
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(a) exposing simultaneously plural single molecule nucleic acid templates immobilized on a surface, each of which is hybridized to a primer, to (i) a polymerase capable of catalyzing nucleotide addition to said primers hybridized to said templates, and (ii) an optically detectable labeled nucleotide that is not a chain terminating nucleotide, under reaction conditions and for a time such that only one nucleotide is added to each said primer by said polymerase to produce plural extended primers incorporating said labeled nucleotide; and (b) optically identifying which of said templates on said surface incorporated said single labeled nucleotide in step (a).
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Specification