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用于在体外高频分离随机单碱基替换的酶促技术。

Enzymatic techniques for the isolation of random single-base substitutions in vitro at high frequency.

作者信息

Abarzúa P, Marians K J

出版信息

Proc Natl Acad Sci U S A. 1984 Apr;81(7):2030-4. doi: 10.1073/pnas.81.7.2030.

DOI:10.1073/pnas.81.7.2030
PMID:6326100
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC345430/
Abstract

A general and efficient method has been developed to generate large numbers of single-base substitution mutations simply and rapidly. A unique f1 phage recombinant DNA cloning vector is described, which contains the phi X174 origin of viral strand DNA synthesis and allows one to direct mutagenesis to any specific segment of DNA. Gapped circular DNA is constructed by annealing viral single-stranded circular DNA [ss(c) DNA] with a mixture of linear duplex DNAs that have had their 3'-OH termini processively digested with Escherichia coli exonuclease III under conditions in which the resulting, newly generated 3'-OH termini present in the various hybrid molecules span the region of interest. Base changes are induced by misincorporation of an alpha-thiodeoxynucleoside triphosphate analog onto this primer-template, followed by DNA repair synthesis. The asymmetric segregation of mutants from wild-type sequences is accomplished by double-stranded replicative form DNA----ss(c) DNA synthesis in vitro, initiated from the phi X174 viral strand origin sequence present on the vector DNA. Mutated ss(c) DNA is screened by the dideoxy chain termination method. In one mutagenesis experiment, 21 independent single-base substitutions were isolated in a 72-nucleotide-long target region. DNA sequence analysis showed that all possible base transversions and transitions were represented.

摘要

已经开发出一种通用且高效的方法，可简单快速地产生大量单碱基取代突变。描述了一种独特的f1噬菌体重组DNA克隆载体，它含有噬菌体X174病毒链DNA合成的起始位点，并能使诱变作用定向于DNA的任何特定片段。通过使病毒单链环状DNA[ss(c)DNA]与线性双链DNA混合物退火构建缺口环状DNA，这些线性双链DNA的3'-OH末端已在大肠杆菌核酸外切酶III作用下进行了连续消化，条件是在各种杂交分子中产生的新生成的3'-OH末端跨越感兴趣的区域。通过将α-硫代脱氧核苷三磷酸类似物错误掺入该引物模板，随后进行DNA修复合成来诱导碱基变化。通过从载体DNA上存在的噬菌体X174病毒链起始序列开始的体外双链复制型DNA----ss(c)DNA合成，实现突变体与野生型序列的不对称分离。通过双脱氧链终止法筛选突变的ss(c)DNA。在一次诱变实验中，在一个72个核苷酸长的靶区域中分离出21个独立的单碱基取代。DNA序列分析表明，所有可能的碱基颠换和转换都有出现。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2fc5/345430/0dc980aa050e/pnas00608-0110-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2fc5/345430/a1bb20f075ae/pnas00608-0109-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2fc5/345430/5d4f13199c22/pnas00608-0109-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2fc5/345430/ccd5bab85fb5/pnas00608-0109-c.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2fc5/345430/0dc980aa050e/pnas00608-0110-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2fc5/345430/a1bb20f075ae/pnas00608-0109-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2fc5/345430/5d4f13199c22/pnas00608-0109-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2fc5/345430/ccd5bab85fb5/pnas00608-0109-c.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2fc5/345430/0dc980aa050e/pnas00608-0110-a.jpg

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本文引用的文献

1
Gap misrepair mutagenesis: efficient site-directed induction of transition, transversion, and frameshift mutations in vitro.缺口错配诱变：体外高效定点诱导转换、颠换和移码突变
Proc Natl Acad Sci U S A. 1982 Mar;79(5):1588-92. doi: 10.1073/pnas.79.5.1588.
2
Size classes of products synthesized processively by two subassemblies of Escherichia coli DNA polymerase III holoenzyme.由大肠杆菌DNA聚合酶III全酶的两个亚组件连续合成的产物的大小类别。
J Biol Chem. 1982 May 25;257(10):5692-9.
3
Local mutagenesis within deletion loops of DNA heteroduplexes.
Mol Cell Biol. 1986 May;6(5):1663-70. doi: 10.1128/mcb.6.5.1663-1670.1986.
4
Misincorporation by AMV reverse transcriptase shows strong dependence on the combination of template and substrate nucleotides.禽成髓细胞瘤病毒逆转录酶的错误掺入显示出对模板和底物核苷酸组合的强烈依赖性。
Nucleic Acids Res. 1986 Sep 11;14(17):6945-64. doi: 10.1093/nar/14.17.6945.
DNA异源双链体缺失环内的局部诱变
Proc Natl Acad Sci U S A. 1982 Dec;79(23):7214-7. doi: 10.1073/pnas.79.23.7214.
4
Sequence specificity of exonuclease III from E. coli.来自大肠杆菌的核酸外切酶III的序列特异性。
Nucleic Acids Res. 1982 Aug 25;10(16):4845-59. doi: 10.1093/nar/10.16.4845.
5
Deoxynucleoside [1-thio]triphosphates prevent proofreading during in vitro DNA synthesis.脱氧核苷[1-硫代]三磷酸在体外DNA合成过程中阻止校对。
Proc Natl Acad Sci U S A. 1981 Nov;78(11):6734-8. doi: 10.1073/pnas.78.11.6734.
6
Separate requirements for leading and lagging strand DNA synthesis during phi X A protein-dependent RF goes to RF DNA replication in vitro.在体外 phi X A 蛋白依赖的 RF 到 RF DNA 复制过程中，前导链和滞后链 DNA 合成的不同要求。
J Biol Chem. 1981 Dec 25;256(24):13143-51.
7
The dnaN gene codes for the beta subunit of DNA polymerase III holoenzyme of escherichia coli.dnaN基因编码大肠杆菌DNA聚合酶III全酶的β亚基。
Proc Natl Acad Sci U S A. 1981 Sep;78(9):5391-5. doi: 10.1073/pnas.78.9.5391.
8
Amplification of single-strand DNA binding protein in Escherichia coli.大肠杆菌中单链DNA结合蛋白的扩增
Nucleic Acids Res. 1980 Jul 25;8(14):3215-27. doi: 10.1093/nar/8.14.3215.
9
Efficient site-directed mutagenesis by simultaneous use of two primers.通过同时使用两种引物进行高效的定点诱变。
Nucleic Acids Res. 1983 Aug 11;11(15):5103-12. doi: 10.1093/nar/11.15.5103.
10
Studies on the phi X174 gene A protein-mediated termination of leading strand DNA synthesis.关于φX174基因A蛋白介导的前导链DNA合成终止的研究。
J Biol Chem. 1983 Jan 10;258(1):529-37.