Brown D M, Frampton J, Goelet P, Karn J
Gene. 1982 Dec;20(2):139-44. doi: 10.1016/0378-1119(82)90032-4.
We have extended the method of Hu and Messing (Gene 17 (1982) 271-277) to prepare highly radioactive M13 probes suitable for use in RNA-DNA hybridization experiments. Single strands of M13 DNA carrying cloned sequences are rendered partially double-stranded by primed synthesis using a synthetic oligonucleotide primer complementary to a region 5' to the cloning site. The newly synthesized radioactive complementary strand is then covalently cross-linked to the M13 phage DNA by UV irradiation in the presence of 4,5,8-trimethylpsoralen (trioxsalen). Since the cross-linked probe is stable to heat denaturation, and the region of cloned sequence is kept single-stranded, these complexes may be used as strand-specific hybridization probes to detect RNA sequences under conditions which would denature DNA-DNA duplexes.
我们扩展了Hu和Messing（《基因》17卷，1982年，271 - 277页）的方法，制备出适用于RNA - DNA杂交实验的高放射性M13探针。携带克隆序列的M13 DNA单链通过使用与克隆位点5'端区域互补的合成寡核苷酸引物进行引物合成，使其部分双链化。然后，在4,5,8 - 三甲基补骨脂素（三甲沙林）存在的情况下，通过紫外线照射将新合成的放射性互补链与M13噬菌体DNA共价交联。由于交联后的探针热变性稳定，且克隆序列区域保持单链状态，这些复合物可用作链特异性杂交探针，在能使DNA - DNA双链变性的条件下检测RNA序列。
