Determining unknown genomic sequence segments, useful e.g. for identifying integration sites in transgenic plants, that border a known sequence

摘要

Determining, even in a very complex genome, unknown sequence segments (A) that flank a known segment (B), is new. Determining, even in a very complex genome, unknown sequence segments (A) that flank a known segment (B), is new. The 5'-A is determined by: (a) cleaving genomic DNA with a restriction enzyme (RE); (b) hybridizing cleavage products with a labeled probe (LP), optionally also an unlabeled bridging probe (BP); (c) purifying the single-stranded (ss) product (SSP) that has hybridized; (d) attaching a double-stranded (ds) adapter (Ad) to one end of SSP, optionally also attaching LP to the other end; (e) preparing and amplifying ds-DNA; and (f) sequence analysis of the amplicon. The 3'-A is determined similarly. Independent claims are also included for the following: (a) nucleic acid molecule (I) that comprises a 5'-adapter, unknown genomic sequence, a vector sequence (or known genomic sequence) and a labeled 3'-probe, also similar structures with a 5'-probe and 3'-adapter; (b) solid carrier with (I) attached to it; and (c) 5'- or 3'-labeled and 5'- or 3'-biotinylated probes.