Grundström T, Zenke W M, Wintzerith M, Matthes H W, Staub A, Chambon P
Nucleic Acids Res. 1985 May 10;13(9):3305-16. doi: 10.1093/nar/13.9.3305.
We describe a rapid and efficient microscale method for in vitro site-directed mutagenesis by gene synthesis. Mutants are constructed by "shot-gun ligation" of overlapping synthetic oligonucleotides yielding double stranded synthetic DNA of more than 120 nucleotides in length. The terminal oligonucleotides of the DNA segment to be synthesized are designed to create sticky ends complementary to unique restriction sites of a polylinker present in an M13 vector. The oligonucleotides are hybridized and ligated to the M13 vector without any purification of the synthetic DNA segment. After cloning, about half of the progeny from such shot-gun ligations contained the predicted sequence demonstrating the efficacy of this method for gene synthesis and its potential for the extensive mutational analysis of genes.
我们描述了一种通过基因合成进行体外定点诱变的快速高效的微量方法。突变体通过重叠合成寡核苷酸的“鸟枪法连接”构建，产生长度超过120个核苷酸的双链合成DNA。待合成DNA片段的末端寡核苷酸被设计成产生与M13载体中多克隆位点的独特限制性位点互补的粘性末端。寡核苷酸无需对合成DNA片段进行任何纯化即可与M13载体杂交并连接。克隆后，这种鸟枪法连接产生的后代中约有一半包含预测序列，证明了该方法用于基因合成的有效性及其对基因进行广泛突变分析的潜力。
