Szybalski W
McArdle Laboratory for Cancer Research, The University of Wisconsin, Madison, WI 53706, USA.
Curr Opin Biotechnol. 1997 Feb;8(1):75-81. doi: 10.1016/s0958-1669(97)80161-9.
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.
基因组中众多限制性位点中的仅一个位点受到同源甲基转移酶（MTase）失活作用的特异性保护，从而产生一个独特的切割位点——阿喀琉斯之踵切割（AC）位点。在RecA-AC（即RARE）技术中，这种特异性保护由一种突触复合物提供，该复合物由RecA蛋白、ATP的γ-S类似物以及一段30至60个核苷酸长的寡脱氧核苷酸组成，该寡脱氧核苷酸与包含受保护限制性位点的序列靶向位点互补或相同。在甲基化以及随后去除保护复合物和MTase后，受保护的位点是同源限制性酶唯一切割的位点。两次这样的靶向切割允许从基因组中切除一个独特的DNA片段。最近的进展包括DNA克隆的校准、缺口的定位以及通过脉冲场凝胶电泳确定切除片段的大小，这使得人们能够测量任意两个相邻序列靶向位点之间的距离，范围从几千碱基到10兆碱基，目的是对基因组进行物理图谱绘制。
