Integration of PCR fragments at any specific site within cloning vectors without the use of restriction enzymes and DNA ligase
Integration of PCR fragments at any specific site within cloning vectors without the use of restriction enzymes and DNA ligase
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DOI：
10.2144/01311st05
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发表时间：
2001-07-01
期刊：
影响因子：
2.7
通讯作者：
Schmitz, R
中科院分区：
文献类型：
--
作者：
Geiser, M;Cèbe, R;Schmitz, R
在这里，我们描述了一种使用限制酶插入DNA的方法，该方法提供了一种独特的方法来设计具有精度但没有消化的质粒。该方法允许将PCR片段插入靶质粒中的任何两个核苷酸之间。唯一的要求，必须将放大片段嵌入与计划集成的位点同源的DNA序列之间。此方法是对Quikchange（TM）定向诱变协议的适应。它比现有的克隆策略更简单，适用于新质粒的多平行构建体。我们通过构建质粒来证明其实用性，在该质粒中，我们成功地整合了最高1117 bp的PCR片段。
Here, we describe a method that offers a unique way to engineer plasmids with precision but without digestion using restriction enzymes for the insertion of DNA. The method allows the insertion of PCR fragments in between any two nucleotides within a target plasmid. The only requirement that the amplified fragments must be embedded between DNA sequences homologous to the site in which the integration is planned. This method is an adaptation of the QuikChange (TM) Site-Directed Mutagenesis protocol. It is simpler than the existing cloning strategies and is suitable for multi-parallel constructions of new plasmids. We have demonstrated its utility by constructing plasmids in which we have successfully integrated PCR fragments up to 1117 bp.