Preparations, Compositions, and Methods for Nucleic Acid Sequencing
First Claim
Patent Images
1. A method of sequencing nucleic acids, comprising:
- a) providing a sample comprising target nucleic acids;
b) adding a set of primers to said sample, wherein the set comprises primers having different nucleotide compositions;
c) contacting the target nucleic acids in the sample with the set of primers under conditions that promote hybridization to generate a set of target-primer complexes;
d) initiating template-directed synthesis of nascent nucleic acid strands from only one primer hybridized to each of the target-primer complexes, wherein the nascent nucleic acid strands are complementary to the target nucleic acids;
e) detecting incorporation of nucleotides into the nascent nucleic acid strands, wherein the detecting further identifies nucleotide sequences of the nascent nucleic acid strands; and
f) based upon the results of step e, determining a nucleotide sequence of the target nucleic acids.
1 Assignment
0 Petitions
Accused Products
Abstract
Preparations, compositions and methods of sequencing nucleic acids are provided. The preparations, methods and compositions employ multiple priming sites on a target nucleic acid and typically utilize single molecule sequencing processes to identify sequence portions of the target which are then assembled to obtain the sequence of the target.
-
Citations
39 Claims
-
1. A method of sequencing nucleic acids, comprising:
-
a) providing a sample comprising target nucleic acids; b) adding a set of primers to said sample, wherein the set comprises primers having different nucleotide compositions; c) contacting the target nucleic acids in the sample with the set of primers under conditions that promote hybridization to generate a set of target-primer complexes; d) initiating template-directed synthesis of nascent nucleic acid strands from only one primer hybridized to each of the target-primer complexes, wherein the nascent nucleic acid strands are complementary to the target nucleic acids; e) detecting incorporation of nucleotides into the nascent nucleic acid strands, wherein the detecting further identifies nucleotide sequences of the nascent nucleic acid strands; and f) based upon the results of step e, determining a nucleotide sequence of the target nucleic acids. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24)
-
-
25-31. -31. (canceled)
-
32. A method of generating sequence information for a target nucleic acid, comprising:
-
providing a plurality of nucleic acid templates corresponding to the target nucleic acid; providing multiple primers that anneal to the nucleic acid templates; annealing the multiple primers to multiple locations on the nucleic acid templates; contacting the nucleic acid templates with a single polymerase enzyme that binds at a single primer annealed to one of the nucleic acid templates; performing sequence-by-synthesis reactions to determine nucleotide sequences of the nucleic acid templates; assembling an overall nucleotide sequence of the target nucleic acid from the nucleotide sequences of the nucleic acid templates determined in step d.
-
-
33. The method of claim 0, wherein the plurality of nucleic acid templates corresponding to the target nucleic acid comprises multiple overlapping fragments with nucleotide sequences identical to portions of the target nucleic acid.
-
34. The method of claim 0, wherein the nucleic acid templates are circular nucleic acid templates that are repeatedly processed in the sequence-by-synthesis reactions to generate multiple copies of the nucleotide sequences of the nucleic acid templates for use in step f.
-
35. (canceled)
-
36. The method of claim 0, wherein the nucleic acid templates are linear nucleic acids and the method further comprises repeating steps c and d multiple times to determine multiple nucleotide sequences from each of the nucleic acid templates downstream of multiple single primers annealed to the nucleic acid templates for use in step f.
-
37-38. -38. (canceled)
-
39. A composition for sequencing a target nucleic acid, comprising:
-
a plurality of nucleic acid templates derived from the target nucleic acid; a set of random primers annealed to the plurality of nucleic acid templates; polymerase enzymes individually immobilized at optically resolvable reaction sites on a substrate, each of which is bound to one of the nucleic acid templates at one of the random primers annealed thereto; and a set of different types of differentially labeled nucleotides, each of which carries a label that emits a signal that is specific to its type that is detectable at the optically resolvable reaction sites.
-
Specification