Efficient base determination in sequencing reactions
First Claim
1. A method for identifying a nucleotide at a target detection position of a target sequence comprising a plurality of detection positions, said method comprising:
- (a) providing a surface comprising a plurality of concatemers, wherein each concatemer comprises a plurality of monomers and each monomer comprises;
i) a target domain of said target sequence comprising a first set of target detection positions;
ii) a first adaptor comprising an anchor site;
b) hybridizing a first anchor probe to said anchor site;
c) hybridizing a second anchor probe to a hybridization site that is between the first anchor site and the target domain and contiguous with both, wherein the second anchor probe is fully degenerate and the hybridization site is in the target sequence and;
d) hybridizing a sequencing probe to said first target domain, wherein said sequencing probe comprises;
i) a probe domain complementary to said target domain, wherein said probe domain includes a unique nucleotide at an interrogation position; and
ii) a label;
under conditions wherein, if said unique nucleotide is complementary to a nucleotide at a target detection position, said sequencing probe hybridizes to said concatemer;
e) ligating said anchor probes and said sequencing probe to form a probe ligation product; and
thenf) detecting said probe ligation product, thereby identifying said nucleotide.
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Abstract
The present invention is directed to compositions and methods for nucleic acid identification and detection. Compositions and methods of the present invention include extracting and fragmenting target nucleic acids from a sample, using the fragmented target nucleic acids to produce target nucleic acid templates and subjecting those target nucleic acid templates to amplification methods to form nucleic acid nanoballs. The invention also includes methods of detecting and identifying sequences using various sequencing applications, including sequencing by ligation methods.
150 Citations
29 Claims
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1. A method for identifying a nucleotide at a target detection position of a target sequence comprising a plurality of detection positions, said method comprising:
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(a) providing a surface comprising a plurality of concatemers, wherein each concatemer comprises a plurality of monomers and each monomer comprises; i) a target domain of said target sequence comprising a first set of target detection positions; ii) a first adaptor comprising an anchor site; b) hybridizing a first anchor probe to said anchor site; c) hybridizing a second anchor probe to a hybridization site that is between the first anchor site and the target domain and contiguous with both, wherein the second anchor probe is fully degenerate and the hybridization site is in the target sequence and; d) hybridizing a sequencing probe to said first target domain, wherein said sequencing probe comprises; i) a probe domain complementary to said target domain, wherein said probe domain includes a unique nucleotide at an interrogation position; and ii) a label; under conditions wherein, if said unique nucleotide is complementary to a nucleotide at a target detection position, said sequencing probe hybridizes to said concatemer; e) ligating said anchor probes and said sequencing probe to form a probe ligation product; and
thenf) detecting said probe ligation product, thereby identifying said nucleotide. - 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, 26, 27, 28, 29)
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Specification