SINGLE-SITE ENZYMATIC CLEAVAGE OF YEAST GENOMIC DNA MEDIATED BY TRIPLE HELIX FORMATION

PHYSICAL mapping of chromosomes would be facilitated by methods of breaking large DNA into manageable fragments, or cutting uniquely at genetic markers of interest. Key issues in the design of sequence-specific DNA cleaving reagents are the specificity of binding, the generalizability of the recognition motif, and the cleavage yield. Oligonucleotide-directed triple helix formation is a generalizable motif for specific binding to sequences longer than 12 base pairs within DNA of high complexity 1-3. Studies with plasmid DNA show that triple helix formation can limit the operational specificity of restriction enzymes to endonuclease recognition sequences that overlap oligonucleotide-binding sites 4,5. Triple helix formation, followed by methylase protection, triple helix-disruption, and restriction endonuclease digestion produces near quantitative cleavage at the single overlapping triple helix-endonuclease site 4,5. As a demonstration that this technique may be applicable to the orchestrated cleavage of large genomic DNA, we report the near quantitative single-site enzymatic cleavage of the Saccharomyces cerevisiae genome mediated by triple helix formation. The 340-kilobase yeast chromosome III was cut uniquely at an overlapping homopurine-EcoRI target site 27 base pairs long to produce two expected cleavage products of 110 and 230 kilobases. No cleavage of any other chromosome was detected. The potential generalizability of this technique, which is capable of near quantitative cleavage at a single site in at lease 14 megabase pairs of DNA, could enable selected regions of chromosomal DNA to be isolated without extensive screening of genomic libraries.