RecA-mediated Achilles' heel cleavage

The specific protection of only one of many restriction sites in a genome from inactivation by a cognate methyltransferase (MTase) creates a unique cleavage site - an Achilles' heel cleavage (AC) site. In the RecA-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 identical to the sequence-targeted site in which the protected restriction site is embedded. Upon methylation and the subsequent removal of the protective complex and MTase, the protected site is the only site cut by the cognate restriction enzyme. Two such targeted cuts permit the excision of a unique DNA fragment from the genome. Recent advances include the calibration of DNA clones, the mapping of gaps, and the determination of the sizes of excised fragments by pulsed-field gel electrophoresis, which allows one to measure distances between any two neighboring sequence-targeted sites, in the range of a few kilobases to 10 megabases, with the purpose of physically mapping the genome.