Efficient base determination in sequencing reactions

摘要

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.

主权项

1. A method for identifying a first nucleotide at a detection position of a target sequence comprising a plurality of detection positions, said method comprising:
(a) providing a plurality of nucleic acid constructs, wherein each nucleic acid construct comprises a plurality of monomers and each monomer comprises:
i) a first target domain of said target sequence comprising a first set of target detection positions; andii) 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 sequence to adjacent to and contiguous with the anchor site, wherein said second anchor probe is partly or entirely degenerate and the hybridized second anchor probe can be ligated to the hybridized first anchor probe; d) hybridizing one, two, or three additional anchor probes to the target sequence between the first target domain and the site of hybridization of the second anchor probe;wherein said additional anchor probe(s) are fully degenerate;
e) hybridizing at least a first sequencing probe to said first target domain,wherein said first sequencing probe comprises:i) a first probe domain complementary to said target domain;ii) a unique nucleotide at a first interrogation position; andiii) a label;under conditions wherein said sequencing probe hybridizes to said target domain if said unique nucleotide is complementary to a first nucleotide in said first set of target detection positions;wherein said hybridized first anchor probe, second anchor probe, additional anchor probe(s) and first sequencing probe are hybridized to adjacent sequences and can be ligated together to form a ligation product; and
f) ligating said anchor probes and said sequencing probe to form a probe ligation product; and then g) detecting said probe ligation product thereby identifying said first nucleotide.