Short cycle methods for sequencing polynucleotides
View Patent ↗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.
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.
2. The method of claim 1 , wherein said label is a detectable label.
3. The method of claim 2 , wherein said detectable label is selected from the group consisting of cyanine, rhodamine, fluorescien, coumarin, BODIPY, alexa, and conjugated multi-dyes.
4. The method of claim 1 , wherein said template is individually optically resolvable.
5. The method of claim 1 , wherein said neutralizing step comprises cleaving said label from said nucleotide.
6. The method of claim 1 , wherein in said exposing step said polymerase and/or said labeled nucleotide are present at concentrations such that a single labeled nucleotide is added to said primer.
7. The method of claim 1 , further comprising the step of washing any unincorporated nucleotide prior to step b.
8. The method of claim 5 , wherein said template is attached to a surface.
9. The method of claim 1 , wherein said single labeled nucleotide is a deoxynucleotide selected from the group consisting of dATP, dTTP, dUTP, dCTP, and dGTP.
10. The method of claim 1 , wherein said single-labeled nucleotide is a nucleotide analog.
11. The method of claim 1 , wherein in said exposing step, the reaction conditions regulate the rate at which said single labeled nucleotide is added to said primer so that only one labeled nucleotide is added to said primer in each cycle.
12. A method for simultaneously sequencing plural nucleic acid templates, the method comprising the steps of:
(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).