A GENERAL-METHOD OF INVITRO PREPARATION AND SPECIFIC MUTAGENESIS OF DNA FRAGMENTS - STUDY OF PROTEIN AND DNA INTERACTIONS
A GENERAL-METHOD OF INVITRO PREPARATION AND SPECIFIC MUTAGENESIS OF DNA FRAGMENTS - STUDY OF PROTEIN AND DNA INTERACTIONS
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DOI：
10.1093/nar/16.15.7351
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发表时间：
1988-08-11
影响因子：
14.9
通讯作者：
SAIKI, RK
中科院分区：
文献类型：
--
作者：
HIGUCHI, R;KRUMMEL, B;SAIKI, RK
使用聚合酶链式反应（PCR）可以简单快速地制备特异性的末端标记的DNA片段。这样的片段适用于DNA酶I保护足迹测定、化学测序反应，以及用于产生和分析暂停的RNA聚合酶转录复合物。此外，还描述了在沿着这种PCR产生的片段的长度的任何沿着位置引入特异性突变的一般方法。这些程序，可以避免需要大规模的噬菌体或质粒生长，制备凝胶电泳和分子克隆的筛选，可以促进蛋白质和DNA的序列特异性相互作用的快速研究。还描述了从完成的PCR中去除过量寡核苷酸引物的快速方法。
Specific, end-labeled DNA fragments can be simply and rapidly prepared using the polymerase chain reaction (PCR). Such fragments are suitable for use in DNase I protection footprint assays, chemical sequencing reactions, and for the production and analysis of paused RNA polymerase transcription complexes. Moreover, a general means of introducing a specific mutation at any position along the length of such PCR-generated fragments is described. These procedures, which can circumvent the need for large-scale phage or plasmid growths, preparative gel-electrophoresis and the screening of molecular clones, can facilitate the rapid study of sequence-specific interactions of proteins and DNA. A rapid means of removing excess oligonucleotide primers from completed PCRs is also described.