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一种无需凝胶纯化步骤且适用于自动化的高效PCR诱变策略。 [“purificiation”的更正为“purification”]

An efficient PCR mutagenesis strategy without gel purification [correction of "purificiation"] step that is amenable to automation.

作者信息

Séraphin B, Kandels-Lewis S

机构信息

EMBL, Heidelberg, Germany.

出版信息

Nucleic Acids Res. 1996 Aug 15;24(16):3276-7. doi: 10.1093/nar/24.16.3276.

DOI:10.1093/nar/24.16.3276
PMID:8774913
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC146073/
Abstract

We describe here an improved megaprimer PCR mutagenesis strategy. The cumbersome gel purification step that is usually used can be omitted by appropriately cleaving the first and second DNA templates with restriction enzymes and enzymatically removing remaining primers from the first PCR reaction. We show that this improved procedure is reproducible and highly efficient. Furthermore this method is suitable for automation because all the steps are now carried out in reaction tubes.

摘要

我们在此描述一种改进的大引物PCR诱变策略。通过用限制酶适当切割第一和第二DNA模板，并从第一次PCR反应中酶促去除剩余引物，通常使用的繁琐凝胶纯化步骤可以省略。我们表明，这种改进的方法具有可重复性且效率很高。此外，该方法适用于自动化，因为现在所有步骤都在反应管中进行。

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本文引用的文献

1
Addition of a class IIS enzyme site in the mutagenic primer to improve two-step PCR-based targeted mutagenesis.在诱变引物中添加IIS类酶切位点以改进基于两步PCR的靶向诱变。
Nucleic Acids Res. 1993 Sep 11;21(18):4424-5. doi: 10.1093/nar/21.18.4424.
2
Improved site-directed mutagenesis method using PCR.利用聚合酶链式反应改进的定点诱变方法。
Nucleic Acids Res. 1991 Aug 25;19(16):4558. doi: 10.1093/nar/19.16.4558.
3
Improved primer design for PCR-based, site-directed mutagenesis.用于基于PCR的定点诱变的改进引物设计。
Nucleic Acids Res. 1992 Mar 11;20(5):1147. doi: 10.1093/nar/20.5.1147.
4
An improved thermal cycle for two-step PCR-based targeted mutagenesis.用于基于两步PCR的靶向诱变的改进热循环
Nucleic Acids Res. 1992 Sep 25;20(18):4934. doi: 10.1093/nar/20.18.4934.