Hu N, Messing J
Gene. 1982 Mar;17(3):271-7. doi: 10.1016/0378-1119(82)90143-3.
A novel approach has been developed for the preparation of highly radioactive, strand-specific M13 probes. A universal primer, complementary to the region 5' to the multiple cloning sites of M13mp7, was used to initiate the DNA synthesis of the complementary strand of the M13 sequence downstream from the inserted sequence. The synthesis of the (-) strand, which was labeled with a radioactively labeled precursor, did not proceed to completion so that the inserted sequence was kept single-stranded. Thus, a partially double-stranded probe that had the specificity of this inserted sequence was obtained. As an example for the application of single-stranded specific hybridization probes, an M13mp7 subclone of a zein cDNA clone of maize (A30) was labeled and used in a dot hybridization test to select from the hundreds of M13mp7 subclones of the zein genomic clone, Z4, the sequences complementary to the probe. The specificity of the probe was confirmed by dideoxy chain terminator sequencing experiments.
已开发出一种制备高放射性、链特异性M13探针的新方法。使用与M13mp7多克隆位点5'端区域互补的通用引物，启动插入序列下游M13序列互补链的DNA合成。用放射性标记的前体标记的（-）链合成未完成，以使插入序列保持单链状态。因此，获得了具有该插入序列特异性的部分双链探针。作为单链特异性杂交探针应用的一个例子，对玉米醇溶蛋白cDNA克隆（A30）的M13mp7亚克隆进行标记，并用于点杂交试验，以从玉米醇溶蛋白基因组克隆Z4的数百个M13mp7亚克隆中筛选出与探针互补的序列。通过双脱氧链终止测序实验证实了探针的特异性。
