Mandecki W
Proc Natl Acad Sci U S A. 1986 Oct;83(19):7177-81. doi: 10.1073/pnas.83.19.7177.
A DNA double-strand break can be efficiently repaired in Escherichia coli if an oligodeoxyribonucleotide is provided to direct the repair. The oligonucleotide must be at least 20 residues long and have a sequence identical to sequences flanking the break. The phenomenon can be used to introduce defined mutations into DNA in the area of a double-strand break. To obtain mutants, the oligonucleotide that carries a mutation and the denatured linearized plasmid DNA are introduced into E. coli by transformation. No enzymatic manipulation in vitro is required. The mutants can constitute up to 98% of the total number of transformants obtained. The efficiency of mutagenesis decreases as the distance between the mutation and the plasmid cleavage site increases. The universality of the method was tested by introducing mutations into four genes, using four plasmids and three E. coli strains, as well as eight restriction enzymes to linearize DNA. Several models of the oligonucleotide-directed DNA double-strand break repair are discussed.
如果提供一个寡脱氧核糖核苷酸来指导修复，DNA双链断裂在大肠杆菌中可以得到有效修复。该寡核苷酸必须至少20个残基长，并且具有与断裂侧翼序列相同的序列。这种现象可用于在双链断裂区域的DNA中引入特定突变。为了获得突变体，将携带突变的寡核苷酸和变性的线性化质粒DNA通过转化引入大肠杆菌。无需体外酶促操作。突变体可占所获得转化体总数的98%。随着突变与质粒切割位点之间距离的增加，诱变效率降低。通过使用四个质粒、三个大肠杆菌菌株以及八种限制酶线性化DNA，将突变引入四个基因，测试了该方法的通用性。讨论了几种寡核苷酸指导的DNA双链断裂修复模型。
