27] Triple helix-mediated single-site enzymatic cleavage of megabase genomic DNA

Abstract

This chapter explains the triple helix-mediated single-site enzymatic cleavage of megabase genomic DNA. Oligonucleotide-directed triple helix formation is a generalizable chemical approach for the recognition and cleavage of a single target site within several megabase pairs of duplex genomic DNA. The generalizability of triple helix-mediated enzymatic cleavage affords high specificity that can be readily customized to unique genetic markers without artificial insertion of a target sequence. Extensive sequencing to identify target sites could be avoided by using degenerate oligonucleotides to screen genetic markers for overlapping triple helix/ endonuclease sites. The potential generalizability of triple helix-mediated Achilles' heel cleavage, a technique capable of near-quantitative cleavage at a single site in at least 14 megabase pairs of DNA, could assist in physical mapping of chromosomal DNA and expedite isolation of DNA segments linked to the disease.