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

A method of capturing and amplifying a selected sequence comprising the following steps, in order:

• 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 synthetically-made 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 synthetically-made selection oligonucleotide of step b) as a template to produce an extended support-bound selection primer that comprises a sequence that is complementary to said genomic sequence;
• d) denaturing the extended selection oligonucleotide of step c) from the support-bound selection primer;
• e) hybridizing said extended support-bound selection primer after step d) to a denatured fragmented genome 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;
• f) extending said extended support-bound selection primer using the strand of genomic DNA as a template to produce an extension product that contains a flanking sequence, wherein the flanking sequence is complementary to a sequence that flanks said genomic sequence in the genome;
• and
• g) amplifying said extension product of step f) 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 that comprises the flanking sequence.