RECA-MEDIATED ACHILLES HEEL CLEAVAGE

The specific protection of only one of many restriction sites in a gen ome from inactivation by a cognate methyltransferase (MTase) creates a unique cleavage site - an Achilles' heel cleavage (AC) site. In the R ecA-AC, or RARE, technique, such specific protection is provided by a synaptic complex composed of RecA protein, a gamma-S analog of ATP and a 30-60 nucleotide long oligodeoxynucleotide complementary or identic al to the sequence-targeted site in which the protected restriction si te is embedded. Upon methylation and the subsequent removal of the pro tective complex and MTase, the protected site is the only site cut by the cognate restriction enzyme. Two such targeted cuts permit the exci sion of a unique DNA fragment from the genome. Recent advances include the calibration of DNA clones, the mapping of gaps, and the determina tion of the sizes of excised fragments by pulsed-field gel electrophor esis, which allows one to measure distances between any two neighborin g sequence-targeted sites, in the range of a few kilobases to 10 megab ases, with the purpose of physically mapping the genome.