Nucleic acid sequencing and process
First Claim
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1. A method of determining a first nucleotide at a detection position of a target sequence comprising a plurality of detection positions, said method comprising:
- a) providing an array comprising a plurality of concatemers randomly disposed on a surface, wherein each concatemer comprises a plurality of monomers and each monomer comprises;
i) a target domain of said target sequence comprising a set of target detection positions;
ii) an adaptor comprising a first anchor site;
b) hybridizing a first anchor probe to said first anchor site;
c) hybridizing a fully degenerate second anchor probe to the target sequence, adjacent to said first anchor site, or,hybridizing a partially degenerate second anchor probe to a second anchor site in the adaptor adjacent to the first anchor site, wherein said partially degenerate second anchor probe also hybridizes to the target sequence;
d) hybridizing a sequencing probe to said target domain, wherein said sequencing probe comprises;
i) a first probe domain complementary to said target domain;
ii) a unique nucleotide at a first interrogation position; and
iii) a label;
under conditions wherein if said unique nucleotide is complementary to said first nucleotide, said sequencing probe hybridizes to said target domain;
e) ligating said anchor probes and said sequencing probe; and
f) identifying said first nucleotidewherein said ligating step (e) is driven to between about 20 percent and about 90 percent completion.
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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.
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Citations
21 Claims
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1. A method of determining a first nucleotide at a detection position of a target sequence comprising a plurality of detection positions, said method comprising:
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a) providing an array comprising a plurality of concatemers randomly disposed on a surface, wherein each concatemer comprises a plurality of monomers and each monomer comprises; i) a target domain of said target sequence comprising a set of target detection positions; ii) an adaptor comprising a first anchor site; b) hybridizing a first anchor probe to said first anchor site; c) hybridizing a fully degenerate second anchor probe to the target sequence, adjacent to said first anchor site, or, hybridizing a partially degenerate second anchor probe to a second anchor site in the adaptor adjacent to the first anchor site, wherein said partially degenerate second anchor probe also hybridizes to the target sequence; d) hybridizing a sequencing probe to said target domain, wherein said sequencing probe comprises; i) a first probe domain complementary to said target domain; ii) a unique nucleotide at a first interrogation position; and iii) a label; under conditions wherein if said unique nucleotide is complementary to said first nucleotide, said sequencing probe hybridizes to said target domain; e) ligating said anchor probes and said sequencing probe; and f) identifying said first nucleotide wherein said ligating step (e) is driven to between about 20 percent and about 90 percent completion. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
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Specification