CONSTRUCTION AND VALIDATION OF YEAST ARTIFICIAL CHROMOSOME CONTIG MAPS BY RECA-ASSISTED RESTRICTION-ENDONUCLEASE CLEAVAGE

RecA-assisted restriction endonuclease (RARE) cleavage is an ''Achille s' heel'' approach to restriction mapping whereby a RecA-protein-oligo deoxynucleotide complex protects an individual restriction site from m ethylation, thus limiting subsequent digestion to a single, predetermi ned site, We have used RARE cleavage to cut yeast artificial chromosom es (YACs) at specific EcoRI sites located within or adjacent to sequen ce-tagged sites (STSs), Each cleavage reaction produces two YAC fragme nts whose sizes are a direct measure of the position of the STS in the YAC. In this fashion, we have positioned 45 STSs within a contig of 1 9 independent YACs and constructed a detailed RARE-cleavage map that r epresents 8.4 Mbp of human chromosome 6p21.3-22. By comparing maps of overlapping YACs, we mere able to detect seven internal deletions that ranged from approximate to 75 kbp to approximate to 1 Mbp in size. Th irteen pairs of EcoRI sites were targeted for double RARE cleavage in uncloned total human DNA, The excised fragments, up to 2 Mbp in size, were resolved by pulsed-field gel electrophoresis and were detected by hybridization. In general, the genomic RARE-cleavage results support the YAC-based map. In one case, the distance in uncloned DNA between t he two terminal EcoRI sites of a YAC insert was approximate to 1 Mbp l arger than the YAC itself, indicating a major deletion, The general co ncept of RARE-cleavage mapping as well as its applications and limitat ions are discussed.