Kunkel T A
Proc Natl Acad Sci U S A. 1985 Jan;82(2):488-92. doi: 10.1073/pnas.82.2.488.
Several single-base substitution mutations have been introduced into the lacZ alpha gene in cloning vector M13mp2, at 40-60% efficiency, in a rapid procedure requiring only transfection of the unfractionated products of standard in vitro mutagenesis reactions. Two simple additional treatments of the DNA, before transfection, produce a site-specific mutation frequency approaching 100%. The approach is applicable to phenotypically silent mutations in addition to those that can be selected. The high efficiency, approximately equal to 10-fold greater than that observed using current methods without enrichment procedures, is obtained by using a DNA template containing several uracil residues in place of thymine. This template has normal coding potential for the in vitro reactions typical of site-directed mutagenesis protocols but is not biologically active upon transfection into a wild-type (i.e., ung+) Escherichia coli host cell. Expression of the desired change, present in the newly synthesized non-uracil-containing covalently closed circular complementary strand, is thus strongly favored. The procedure has been applied to mutations introduced via both oligonucleotides and error-prone polymerization. In addition to its utility in changing DNA sequences, this approach can potentially be used to examine the biological consequences of specific lesions placed at defined positions within a gene.
在克隆载体M13mp2的lacZα基因中，已通过一种快速程序引入了几种单碱基取代突变，效率为40%-60%，该程序仅需转染标准体外诱变反应的未分级产物。在转染前对DNA进行两种简单的额外处理，可使位点特异性突变频率接近100%。该方法除了适用于可选择的突变外，还适用于表型沉默突变。通过使用含有几个尿嘧啶残基代替胸腺嘧啶的DNA模板，可获得高效率，比使用当前无富集程序的方法观察到的效率高约10倍。该模板对于定点诱变方案典型的体外反应具有正常的编码潜力，但转染到野生型（即ung+）大肠杆菌宿主细胞中时无生物学活性。因此，强烈有利于在新合成的不含尿嘧啶的共价闭合环状互补链中出现所需的变化。该程序已应用于通过寡核苷酸和易错聚合引入的突变。除了在改变DNA序列方面的效用外，这种方法还可潜在地用于研究基因内特定位置处特定损伤的生物学后果。
