Overlap hybridization screening: isolation and characterization of overlapping DNA fragments surrounding the leu2 gene on yeast chromosome III

Abstract

A set of four plasmids containing overlapping segments comprising a total of about 30 kbp of cloned DNA from chromosome III of yeast (Saccharomyces cerevisiae) has been isolated and characterized by restriction endonuclease analyses and DNA:DNA hybridizations. Colony hybridization was carried out with labeled pYe(leu2)10, a plasmid carrying the yeast leu2 gene, to a bank of bacterial colonies containing recombinant plasmids constructed from the vector ColE1 and random fragments of yeast DNA. This resulted in the detection of two plasmids, pYe11G4 and pYe40C3, with DNA inserts which partially overlap the original cloned segment and contain additional DNA extending in opposite directions on the chromosome. By carrying out a second round of colony hybridization with pYe40C3, the cloned region was further extended in one direction. A region of DNA that is repeated at least ten times in the yeast genome was identified by hybridization of pYe11G4 to an EcoRI digest of total yeast DNA. The procedure described in this paper should allow the isolation of large sections of chromosomes, including non-transcribed regions, surrounding cloned genes.