Patent ID: 6677121
Filing Date: 2004-01-13
Classification: C12Q,Y10S

Abstract:
A method of producing nucleic acid fragments with adaptors, the method comprising(a) incubating a nucleic acid sample with one or more nucleic acid cleaving reagents that cleave at a site offset from the recognition sequence of the nucleic acid cleaving reagent to produce nucleic acid fragments with sticky ends, (b) mixing one or more adaptor-indexers with the nucleic acid sample and covalently coupling the adaptor-indexers to the nucleic acid fragments, wherein each adaptor-indexer has a different sticky end, wherein each sticky end of the adaptor-indexers is compatible with a sticky end generated by the nucleic acid cleaving reagents, wherein both strands of the adaptor-indexers are covalently coupled to the nucleic acid fragments, (c) incubating the nucleic acid sample with one or more second nucleic acid cleaving reagents, wherein at least one of the second nucleic acid cleaving reagents cleaves in the recognition site of the second nucleic acid cleaving reagent, and (d) mixing one or more second adaptors with the nucleic acid sample and covalently coupling the second adaptors to the nucleic acid fragments, wherein each second adaptor has a sticky end, wherein each sticky end of the second adaptors is compatible with a sticky end generated by the second nucleic acid cleaving reagents, whereby nucleic acid fragments comprising an adaptor-indexer and a second adaptor are formed, wherein multiple fragments comprising an adaptor-indexer and a second adaptor are derived from each of a plurality of nucleic acid molecules in the nucleic acid sample.