Gap misrepair mutagenesis: efficient site-directed induction of transition, transversion, and frameshift mutations in vitro.
Gap misrepair mutagenesis: efficient site-directed induction of transition, transversion, and frameshift mutations in vitro.
复制标题
间隙错误修复诱变：体外有效定点诱导过渡、颠换和移码突变。
DOI：
10.1073/pnas.79.5.1588
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发表时间：
1982
影响因子：
11.1
通讯作者：
Botstein,D
中科院分区：
文献类型：
--
作者：
Shortle,D;Grisafi,P;Benkovic,SJ;Botstein,D
短的单链间隙可以通过有限的外切酶作用在封闭的环状DNA分子上预定位置的单链断裂（缺口）上构建。作为DNA聚合酶的高效引物模板，单链间隙可以在体外修复以再生完整的DNA双链。在本报告中描述了两种体外反应方案，它们在修复间隙期间产生高频率的错误（“错误修复”），从而允许有效地恢复限于原始间隙或其附近的核苷酸序列的突变。在第一种错误修复方案中，由于遗漏了四种脱氧核苷三磷酸中的一种，从而刺激了核苷酸错误结合；由于反应混合物中过量的DNA连接酶的存在，误结合物被捕获。第二种错误修复方案涉及抗切除α -硫代磷酸核苷酸的错误结合，随后在所有四种常规脱氧核苷三磷酸存在的情况下填补间隙。当应用于小质粒pBR322上几个独特的限制性位点之一的短间隙时，两种间隙错误修复方法都在目标限制性位点内产生了高频率的突变（6- 42%）。鉴定出的大部分序列变化是碱基置换；颠倒和过渡大致相等。其余的序列变化是一个碱基对的插入和一到四个碱基对的缺失。
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.