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 period of time such that on average one to two nucleotides are added to said primer by said polymerase to produce an extended primer incorporating said labeled nucleotide;
(b) washing away unincorporated nucleotides;
(c) identifying said labeled nucleotides incorporated in step (a);
(d) neutralizing label in any incorporated nucleotide;
(e) repeating steps a, b, c, and d at least once; and
(f) determining a sequence of said template based upon the order of incorporation of said labeled nucleotides,
wherein said method does not utilize a blocking moiety.
2. The method of claim 1 , wherein said period of time is concluded by washing away said polymerization agent.
3. The method of any of claim 1 , wherein said period is no more than 5 half-lives of said incorporation reactions.
4. The method of claim 1 , wherein said period is no more than 4 half-lives of said incorporation reactions.
5. The method of claim 1 , wherein said period is no more than 3 half-lives of said incorporation reactions.
6. The method of claim 1 , wherein said period is no more than 2 half-lives of said incorporation reactions.
7. The method of claim 1 , wherein said period is no more than 1 half-lives of said incorporation reactions.
8. The method of claim 1 , wherein said period is no more than 0.5 half-lives of said incorporation reactions.
9. The method of claim 1 , wherein said period permits less than 5% chance of incorporation of more than two of said nucleotides into said complementary strand.
10. The method of claim 1 , wherein steps a, b, c, and d are repeated at least 40 times.
11. The method of claim 1 , wherein said label is an optically detectable label.
12. The method of claim 11 , wherein said optically detectable label is selected from the group consisting of cyanine, rhodamine, fluorescien, coumarin, BODIPY, alexa, and conjugated multi-dyes.
13. The method of claim 1 , wherein said template is individually optically resolvable.
14. The method of claim 1 , wherein said neutralizing step comprises cleaving said label from said nucleotide.
15. The method of claim 1 , wherein said template is attached to a surface.
16. The method of claim 1 , wherein said labeled nucleotide is a deoxynucleotide selected from the group consisting of dATP, dTTP, dUTP, dCTP, and dGTP.
17. The method of claim 1 , wherein said labeled nucleotide is a nucleotide analog.