PHYSICAL CALIBRATION OF YEAST ARTIFICIAL CHROMOSOME CONTIG MAPS BY RECA-ASSISTED RESTRICTION-ENDONUCLEASE (RARE) CLEAVAGE

Clone-based genome maps can be constructed by determining the presence or absence of sequence-tagged sites (STSs) in a redundant collection of yeast artificial chromosome clones (YACs). While STS-content mappin g has proven to be an effective means of ordering clone ends and STSs along chromosomes, the exact physical map positions of these landmarks are not determined. This fundamental weakness can be overcome by RecA -assisted restriction endonuclease (RARE) cleavage, a method that expl oits the binding specificity on duplex DNA of a RecA-protein-oligodeox ynucleotide complex to enhance the cleavage specificity of a restricti on endonuclease. This technique allows selective cleavage at individua l members of a large set of restriction sites. RARE-cleavage mapping w as applied to a contig comprising 5 overlapping YACs spanning 580 kb o n human chromosome 14. An STS-content map comprising 10 YAC-end specif ic STSs and one internal STS was constructed. RARE cleavage was perfor med on 2 YACs that span the entire contig at the EcoRI sites defining the vector-insert junctions of all 5 YACs, as well as at a HhaI site w ithin the STS that was initially used to screen the YAC library for th e clones in the contig. The sizes of the RARE-cleavage fragments were measured by pulsed-field gel electrophoresis and used to convert the S TS-content map into a true physical map that indicates precise positio ns of clone ends and STSs. (C) 1994 Academic Press, Inc.