Direct isolation of human BRCA2 gene by transformation-associated recombination in yeast

Direct isolation of human BRCA2 gene by transformation-associated recombination in yeast

^*Laboratories of Molecular Genetics and ^‡Molecular Carcinogenesis, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709

Abstract

Mutant forms of the BRCA2 gene contribute significantly to hereditary breast cancer. Isolation of the normal and mutant forms of the BRCA2 gene with its natural promoter would greatly facilitate analysis of the gene and its contribution to breast cancer. We have accomplished the direct isolation of the 90-kb gene from total human DNA by transformation-associated recombination in yeast using a small amount of 5′ and 3′ BRCA2 sequence information. Because the entire isolation procedure of a single chromosomal gene could be accomplished in approximately 2 weeks, the transformation-associated recombination cloning approach is readily applicable to studies of chromosome alterations and human genetic diseases.

Footnotes

↵ † To whom reprint requests should be addressed at: Laboratory of Molecular Genetics, National Institute on Environmental Health Sciences, Box 12233, Research Triangle Park, NC 27709. e-mail: larionov{at}niehs.nih.gov.
Alexander Varshavsky, California Institute of Technology, Pasadena, CA
ABBREVIATIONS:

TAR,

transformation-associated recombination;

ARS,

autonomously replicating sequences;

TAFE,

transverse alternating field electrophoresis;

BAC and YAC,

bacterial and yeast artificial chromosome, respectively