Weiher H, Schaller H
Proc Natl Acad Sci U S A. 1982 Mar;79(5):1408-12. doi: 10.1073/pnas.79.5.1408.
A method for highly efficient segment-specific mutagenesis is described. The method uses as target for sodium bisulfite mutagenesis the DNA single strands of a DNA restriction fragment that had been separated by cloning into base-complementary regions of a pair of phage fd vectors. After repair synthesis in vitro, the mutagenized DNA fragment is recovered by cloning into a nonmutated plasmid vector and analyzed for sequence and by functional tests. By using this method, the nucleotide sequence of a 109-base pair restriction fragment containing the lac promoter/operator from Escherichia coli was extensively modified. More than 90% of the 235 isolates obtained showed a change in phenotype; all of 22 analyzed for their nucleotide sequence were found to carry multiple C leads to T point mutations in up to 60% of the possible target positions. Nevertheless, few isolates showed major changes in promoter activity relative to the nonmutated promoter element, which indicates a high degree of flexibility in the promoter sequence.
描述了一种高效的片段特异性诱变方法。该方法将通过克隆到一对噬菌体fd载体的碱基互补区域而分离的DNA限制性片段的DNA单链用作亚硫酸氢钠诱变的靶标。在体外进行修复合成后,通过克隆到未突变的质粒载体中回收诱变的DNA片段,并进行序列分析和功能测试。通过使用该方法,对来自大肠杆菌的包含lac启动子/操纵子的109个碱基对的限制性片段的核苷酸序列进行了广泛修饰。在获得的235个分离株中,超过90%表现出表型变化;对其核苷酸序列进行分析的22个分离株中,所有分离株在多达60%的可能靶位点都携带多个C突变为T的点突变。然而,相对于未突变的启动子元件,很少有分离株显示出启动子活性的主要变化,这表明启动子序列具有高度的灵活性。