Sena E P, Zarling D A
Cell and Molecular Biology Laboratory, SRI International, Menlo Park, California 94025.
Nat Genet. 1993 Apr;3(4):365-72. doi: 10.1038/ng0493-365.
A new in vitro hybridization reaction targets two short complementary RecA protein-coated DNA probes to homologous sequences at any position in a linear duplex DNA molecule. Stable hybrids are obtained after RecA protein removal when both complementary probe strands are present in a four-stranded hybrid, but not when one probe strand is present in a three-stranded hybrid. In four-stranded hybrids with one probe strand biotinylated and the other radiolabelled, the deproteinized hybrids can be isolated and detected by affinity capture on streptavidin-coated magnetic beads. RecA-mediated targeting of complementary biotinylated DNA probe strands allows the affinity capture of 48.5-kilobase duplex lambda genomic DNA. These reactions provide a means of isolating any desired duplex gene or chromosomal DNA fragment.
一种新的体外杂交反应将两个短的互补RecA蛋白包被的DNA探针靶向线性双链DNA分子中任意位置的同源序列。当两条互补探针链存在于四链杂交体中时，去除RecA蛋白后可获得稳定的杂交体，但当一条探针链存在于三链杂交体中时则不然。在一条探针链生物素化而另一条探针链放射性标记的四链杂交体中，脱蛋白的杂交体可通过在链霉亲和素包被的磁珠上进行亲和捕获来分离和检测。RecA介导的互补生物素化DNA探针链靶向使得能够亲和捕获48.5千碱基的双链λ基因组DNA。这些反应提供了一种分离任何所需双链基因或染色体DNA片段的方法。
