COINCIDENCE PAINTING - A RAPID METHOD FOR CLONING REGION-SPECIFIC DNA-SEQUENCES

We have developed a novel coincidence cloning strategy, termed Coincid ence Painting, which enables the rapid generation of large numbers of region specific sequences. Coincidence Painting utilises Degenerate Ol igonucleotide Primed PCR (DOP-PCR) amplification of flow sorted deriva tive translocation chromosomes. The PCR products are hybridised in sit u onto specific flow sorted chromosomes for coincident sequence select ion. Eluted and reamplified material is then cloned using a novel inse rt end revelation and ligation technique. Cloned inserts range in size from 150 - 1300 bps of which approximately 54% appear to be single co py sequences. The cloning method permits the excision of vector free p robe for library hybridisation screening and the small insert size fac ilitates analysis for the generation of sequence tagged sites (STSs). We have used such clones successfully for YAC screening by PCR and for cosmid screening by filter hybridisation. This new methodology should allow the rapid saturation with probes of regions defined by specific translocation breakpoints.