Dalbadie-McFarland G, Cohen L W, Riggs A D, Morin C, Itakura K, Richards J H
Proc Natl Acad Sci U S A. 1982 Nov;79(21):6409-13. doi: 10.1073/pnas.79.21.6409.
We have used oligonucleotide-directed mutagenesis to make a specific change in the beta-lactamase (EC 3.5.2.6) (ampicillin resistance) gene of the plasmid pBR322. Evidence suggests that the active site for this enzyme may include a serine-threonine dyad (residues 70 and 71). By priming in vitro DNA synthesis with a chemically synthesized 16-base oligodeoxyribonucleotide, we have inverted the Ser-Thr dyad to Thr-Ser and thereby generated a mutant with an ampicillin-sensitive phenotype. This "double-mismatch" method is relatively simple and also very general because detection of mutants is at the level of DNA and involves only colony hybridization. Accordingly, the procedure can be applied to any DNA sequence and does not depend on the phenotype of the mutant.
我们利用寡核苷酸定向诱变技术，对质粒pBR322的β-内酰胺酶（EC 3.5.2.6）（氨苄青霉素抗性）基因进行了特定改变。有证据表明，该酶的活性位点可能包括一个丝氨酸-苏氨酸二元组（第70和71位氨基酸残基）。通过用化学合成的16碱基寡脱氧核糖核苷酸引发体外DNA合成，我们将丝氨酸-苏氨酸二元组颠倒为苏氨酸-丝氨酸，从而产生了具有氨苄青霉素敏感表型的突变体。这种“双错配”方法相对简单且通用性很强，因为突变体的检测是在DNA水平上进行的，仅涉及菌落杂交。因此，该方法可应用于任何DNA序列，且不依赖于突变体的表型。
