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一种用于克隆DNA区域特异性和链特异性诱变的快速高效方法。

A rapid and efficient method for region- and strand-specific mutagenesis of cloned DNA.

作者信息

Everett R D, Chambon P

出版信息

EMBO J. 1982;1(4):433-7. doi: 10.1002/j.1460-2075.1982.tb01187.x.

DOI:10.1002/j.1460-2075.1982.tb01187.x
PMID:6329679
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC553064/
Abstract

The single-stranded viral DNA of an M13 phage recombinant containing the early promoter region of SV40 was hybridized with linear, double-stranded replicative form DNA of a related M13 phage containing a short deletion in the cloned SV40 sequence. The heteroduplexes formed between these DNA molecules contained a short, defined single-stranded region in an otherwise duplex molecule. These heteroduplexes were treated with sodium bisulphite to deaminate exposed unpaired cytosines to uracil residues. The single-stranded region was filled in with DNA polymerase I, which incorporates adenine opposite the mutated uracils, and the DNA then transfected into the M13 host JM103 . Viral DNA from the resultant plaques was used for the rapid dideoxy-DNA sequencing procedure; all of the plaques studied contained point mutations within the desired area. This method allows the very rapid and efficient generation of region-directed point mutants which can be quickly sequenced.

摘要

含有SV40早期启动子区域的M13噬菌体重组体的单链病毒DNA与相关M13噬菌体的线性双链复制形式DNA杂交，该相关M13噬菌体在克隆的SV40序列中有一个短缺失。这些DNA分子之间形成的异源双链体在其他双链分子中包含一个短的、明确的单链区域。用亚硫酸氢钠处理这些异源双链体，使暴露的未配对胞嘧啶脱氨基成为尿嘧啶残基。用DNA聚合酶I填补单链区域，该酶在突变的尿嘧啶相对位置掺入腺嘌呤，然后将DNA转染到M13宿主JM103中。来自所得噬菌斑的病毒DNA用于快速双脱氧DNA测序程序；所有研究的噬菌斑在所需区域内都含有点突变。这种方法能够非常快速和高效地产生可快速测序的区域定向点突变体。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/67a8/553064/3ccbf684e3c2/emboj00296-0043-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/67a8/553064/3ccbf684e3c2/emboj00296-0043-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/67a8/553064/3ccbf684e3c2/emboj00296-0043-a.jpg

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本文引用的文献

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The effect of bisulfite modification on the template activity of DNA for DNA polymerase I.亚硫酸氢盐修饰对DNA聚合酶I模板活性的影响。
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Nucleic Acids Res. 1982 Sep 11;10(17):5161-71. doi: 10.1093/nar/10.17.5161.
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Targeted mutagenesis in vitro: lac repressor mutations generated using AMV reverse transcriptase and dBrUTP.体外定向诱变：使用禽成髓细胞瘤病毒逆转录酶和二溴尿苷三磷酸生成的乳糖阻遏物突变
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