Weiner M P, Felts K A, Simcox T G, Braman J C
Stratagene Cloning Systems, La Jolla, CA 92037.
Gene. 1993 Apr 15;126(1):35-41. doi: 10.1016/0378-1119(93)90587-s.
A general solid-phase method for the site-directed mutagenesis of double-stranded DNA (dsDNA) is described. Plasmid DNA is linearized using either a restriction endonuclease (ENase) or the RecA-assisted ENase or RecA-AC cleavage method. Alternatively, PCR may be used to generate linear dsDNA. One or both strands of the DNA is biotinylated and attached to a solid support, and the DNA strands are separated using 0.2 M NaOH. An extension oligodeoxyribonucleotide (oligo) and a single or multiple oligo(s) containing the desired mutation(s) are annealed to one of the bound DNA strands and used to initiate the synthesis of a complementary strand by a nonstrand-displacing DNA polymerase. The in vitro synthesized strand incorporating the desired alteration(s) is melted off of the support and recircularized using one of several types of bridging oligos, DNA ligase, and a DNA polymerase and transformed into the host. Greater than 90% mutagenic efficiency has been obtained using this method.
本文描述了一种用于双链DNA（dsDNA）定点诱变的通用固相方法。使用限制性内切酶（ENase）或RecA辅助的ENase或RecA-AC切割方法使质粒DNA线性化。或者，也可以使用PCR来生成线性dsDNA。将DNA的一条或两条链进行生物素化并连接到固相支持物上，然后用0.2 M NaOH分离DNA链。将延伸寡脱氧核糖核苷酸（oligo）和包含所需突变的一个或多个oligo与结合的DNA链之一退火，并用于通过非链置换DNA聚合酶启动互补链的合成。将掺入所需改变的体外合成链从支持物上熔解下来，并使用几种类型的桥接oligo、DNA连接酶和DNA聚合酶之一进行环化，然后转化到宿主中。使用该方法已获得大于90%的诱变效率。
