Direct capture, amplification and sequencing of target DNA using immobilized primers

摘要

Certain embodiments provide a method for capturing a genomic fragment. The method may comprise: obtaining a substrate comprising a first population of surface-bound oligonucleotides and a second population of surface-bound oligonucleotides; hybridizing a first member of the first population of surface-bound oligonucleotides to a selection oligonucleotide comprising a region that hybridizes with the first member and a region that contains a genomic sequence; extending the first member of the first population of surface-bound oligonucleotides to produce a support-bound selection primer that comprises a sequence that is complementary to the genomic sequence; hybridizing the support-bound selection primer to a nucleic acid fragment comprising the genomic sequence; extending the support-bound selection primer to produce an extension product that contains a sequence that flanks the genomic sequence, e.g., in a genome; and amplifying the extension product on the substrate.

主权项

1. A method of capturing and amplifying a selected sequence comprising:
a) obtaining a substrate comprising a first population of surface-bound oligonucleotides and a second population of surface-bound oligonucleotides, wherein the members of said first and second populations of surface-bound oligonucleotides are attached to the same substrate but not spatially addressed on said substrate; b) hybridizing a first member of said first population of surface-bound oligonucleotides to a selection oligonucleotide comprising a region that hybridizes with said first member and a region that contains a genomic sequence, c) extending said first member of said first population of surface-bound oligonucleotides using the hybridized selection oligonucleotide of (b) as a template to produce an extended support-bound selection primer that comprises a sequence that is complementary to said genomic sequence; d) hybridizing said extended support-bound selection primer of step c) to denatured fragmented genomic DNA to produce a duplex comprising said extended support-bound selection primer and a strand of genomic DNA that comprises said genomic sequence, wherein, in the duplex, the strand of genomic DNA is at least 100 bases in length and the 3′ end of the extended support-bound selection primer is extendible using the strand of genomic DNA as a template; e) extending said extended support-bound selection primer using the strand of genomic DNA as a template to produce an extension product that contains the complement of a sequence that flanks said genomic sequence; and f) amplifying said extension product of step e) on said substrate by bridge PCR using unextended members of said first and second populations of surface-bound oligonucleotides of step a), to produce a PCR product.