Shortle D, Grisafi P, Benkovic S J, Botstein D
Proc Natl Acad Sci U S A. 1982 Mar;79(5):1588-92. doi: 10.1073/pnas.79.5.1588.
Short single-stranded gaps can be constructed by limited exonuclease action at single-stranded breaks (nicks) placed at predetermined sites on closed circular DNA molecules. As efficient primer-templates for DNA polymerase, single-stranded gaps can be repaired in vitro to regenerate an intact DNA duplex. In this report two in vitro reaction schemes are described that produce a high frequency of errors during repair ("misrepair") of gaps and thereby allow the efficient recovery of mutations limited to the nucleotide sequence at or near the original gap. In the first of these misrepair schemes, nucleotide misincorporations are stimulated by omission of one of the four deoxynucleoside triphosphates; the misincorporations are trapped by the presence of excess DNA ligase in the reaction mixture. The second misrepair scheme involves the misincorporation of an excision-resistant alpha-thiophosphate nucleotide, followed by gap filling in the presence of all four conventional deoxynucleoside triphosphates. When applied to short gaps constructed at one of several unique restriction sites on the small plasmid pBR322, both gap misrepair methods yielded mutations within the targeted restriction site at high frequency (6--42%). A majority of the sequence changes identified were base substitutions; transversions and transitions are approximately equally represented. The remaining sequence changes were an insertion of a single base pair and deletions of one to four base pairs.
短单链缺口可通过在闭环DNA分子上预定位点处的单链断裂（切口）进行有限的核酸外切酶作用来构建。作为DNA聚合酶的有效引物模板，单链缺口可在体外修复以再生完整的DNA双链体。在本报告中，描述了两种体外反应方案，它们在缺口修复（“错配修复”）过程中产生高频率的错误，从而允许有效地回收限于原始缺口处或其附近核苷酸序列的突变。在这些错配修复方案中的第一种中，通过省略四种脱氧核苷三磷酸之一来刺激核苷酸错掺入；反应混合物中过量DNA连接酶的存在会捕获错掺入。第二种错配修复方案涉及抗切除的α-硫代磷酸核苷酸的错掺入，然后在所有四种常规脱氧核苷三磷酸存在下进行缺口填充。当应用于在小质粒pBR322上几个独特限制位点之一构建的短缺口时，两种缺口错配修复方法都在目标限制位点内产生了高频率（6%-42%）的突变。鉴定出的大多数序列变化是碱基替换；颠换和转换的出现频率大致相等。其余的序列变化是单个碱基对的插入和一至四个碱基对的缺失。
