Selective Cleavage of Human DNA: RecA-Assisted Restriction Endonuclease (RARE) Cleavage

Abstract

Current methods for sequence-specific cleavage of large segments of DNA are severely limited because of the paucity of possible cleavage sites. A method is described whereby any Eco RI site can be targeted for specific cleavage. The technique is based on the ability of RecA protein from Escherichia coli to pair an oligonucleotide to its homologous sequence in duplex DNA and to form a three-stranded complex. This complex is protected from Eco RI methylase; after methylation and RecA protein removal, Eco RI restriction enzyme cleavage was limited to the site previously protected from methylation. When pairs of oligonucleotides are used, a specific fragment can be cleaved out of genomes. The method was tested on lambda phage, Escherichia coli, and human DNA. Fragments exceeding 500 kilobases in length and yields exceeding 80 percent could be obtained.