Fritz H J, Hohlmaier J, Kramer W, Ohmayer A, Wippler J
Max-Planck-Institut für Biochemie, Abteilung Zellbiologie, Martinsried bei München, FRG.
Nucleic Acids Res. 1988 Jul 25;16(14B):6987-99. doi: 10.1093/nar/16.14.6987.
The gapped duplex DNA approach to oligonucleotide-directed construction of mutations (Kramer et al. 1984, Nucl. Acids Res. 12, 9441-9456) has been developed further. A procedure is described that makes in vitro DNA polymerase/DNA ligase reactions dispensable. Direct transfection of host bacteria with gdDNA molecules of recombinant phage M13 plus mutagenic oligonucleotide results in marker yields in excess of 50% (gap size 1640 nucleotides). An important feature incorporated into the mutagenic oligonucleotide is the presence of one or two internucleotidic phosphorothioate linkages immediately adjacent to the 5'-terminus. Automated preparation and biochemical properties of such compounds are described as well as their performance in oligonucleotide-directed mutagenesis. A systematic study of the following parameters influencing marker yield is reported: Gap size, length of oligonucleotide, chemical nature of oligonucleotide termini and heatshock temperature during transformation.
用于寡核苷酸定向构建突变的缺口双链DNA方法（Kramer等人，1984年，《核酸研究》12卷，9441 - 9456页）已得到进一步发展。本文描述了一种无需体外DNA聚合酶/DNA连接酶反应的方法。用重组噬菌体M13的gdDNA分子加上诱变寡核苷酸直接转染宿主细菌，可使标记产量超过50%（缺口大小为1640个核苷酸）。诱变寡核苷酸中纳入的一个重要特征是在紧邻5'端的位置存在一个或两个核苷酸间硫代磷酸酯键。本文描述了此类化合物的自动化制备、生化特性及其在寡核苷酸定向诱变中的性能。报告了对影响标记产量的以下参数的系统研究：缺口大小、寡核苷酸长度、寡核苷酸末端的化学性质以及转化过程中的热休克温度。
