High throughput genome sequencing on DNA arrays
The present invention is directed to methods and compositions for acquiring nucleotide sequence information of target sequences using adaptors interspersed in target polynucleotides. The sequence information can be new, e.g. sequencing unknown nucleic acids, re-sequencing, or genotyping. The invention preferably includes methods for inserting a plurality of adaptors at spaced locations within a target polynucleotide or a fragment of a polynucleotide. Such adaptors may serve as platforms for interrogating adjacent sequences using various sequencing chemistries, such as those that identify nucleotides by primer extension, probe ligation, and the like. Encompassed in the invention are methods and compositions for the insertion of known adaptor sequences into target sequences, such that there is an interruption of contiguous target sequence with the adaptors. By sequencing both “upstream” and “downstream” of the adaptors, identification of entire target sequences may be accomplished.
1 - 42 . (canceled)
43 . A method of inserting multiple adaptors in a target nucleic acid comprising:
a) ligating a first adaptor to one terminus of said target nucleic acid to form a first linear construct, wherein said adaptor comprises a recognition site for a first restriction enzyme;
b) circularizing first linear construct to create a first circular polynucleotide;
c) cleaving said first circular polynucleotide with said first restriction enzyme to form a second linear construct, wherein said first restriction enzyme is able to bind to said recognition site within said first adaptor;
d) ligating a second adaptor to said second linear construct to form a third linear construct, wherein said second adaptor comprises a recognition site for a second restriction enzyme;
e) circularizing said third linear construct to create a second circular polynucleotide.
44 - 82 . (canceled)