Kadonaga J T, Knowles J R
Nucleic Acids Res. 1985 Mar 11;13(5):1733-45. doi: 10.1093/nar/13.5.1733.
A simple and efficient procedure for the generation of random GC to AT transition mutations in a specific DNA segment is described. A restriction fragment is inserted in each orientation into an M13 vector, single-stranded virion DNA from each recombinant phage is treated with methoxylamine, and, after reannealing of the mutagenized strands, a double-stranded restriction fragment is obtained. This methoxylamine-derivatized DNA segment is then joined with linearized M13 RF DNA, competent E. coli is transfected, and mutations are directly identified by sequencing of the phage DNA. Using this technique, single and double nucleotide substitutions were generated at a frequency greater than 50% in a 56-base pair segment of the signal codons of the TEM beta-lactamase.
本文描述了一种在特定DNA片段中产生随机GC到AT转换突变的简单高效方法。将一个限制性片段以每种方向插入到M13载体中，来自每个重组噬菌体的单链病毒体DNA用甲氧基胺处理，并且在诱变链重新退火后，获得双链限制性片段。然后将这个甲氧基胺衍生化的DNA片段与线性化的M13 RF DNA连接，转染感受态大肠杆菌，并通过对噬菌体DNA进行测序直接鉴定突变。使用该技术，在TEMβ-内酰胺酶信号密码子的56个碱基对片段中，单核苷酸和双核苷酸取代的产生频率大于50%。
