Cloning and assembly of PCR products using modified primers and DNA repair enzymes
Cloning and assembly of PCR products using modified primers and DNA repair enzymes
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DOI：
10.2144/97235st01
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发表时间：
1997-11-01
期刊：
影响因子：
2.7
通讯作者：
Bennett, GN
中科院分区：
文献类型：
--
作者：
Watson, DE;Bennett, GN
我们提出了一种创建可连接 3' 突出端的方法，方法是在 PCR 引物的特定位置掺入修饰碱基尿嘧啶，随后用 DNA 修饰酶尿嘧啶 DNA 糖基化酶处理，然后用 T4 核酸内切酶 V 或人无嘌呤/无嘧啶核酸内切酶 1 处理。在这项研究中，我们描述了将指定氯霉素抗性基因的片段克隆到SacI 载体位点。为了进一步测试该方法，通过 PCR 扩增了 lacZ 基因的三个片段，并在用 DNA 修饰酶处理后，将正确定向的片段连接到 SacI 切割的质粒中。使用所描述的方法，我们能够将 PCR 产物组装成适当的结构。
We present a method for the creation of ligatable 3' overhangs by the incorporation of a modified base, uracil, at a specific position in the PCR primer and subsequent treatment with the DNA-modifying enzyme uracil DNA glycosylase and then either T4 endonuclease V or human apurinic/apyrimidinic endonuclease 1. In this study, we describe the cloning of a fragment specifying the chloramphenicol-resistance gene into a SacI vector site. To further test this method, three segments of the lacZ gene were amplified by PCR, and after treatment with the DNA-modifying enzymes, the properly oriented segments were ligated into a SacI-cleaved plasmid. Using the methods described, we were able to assemble PCR products into appropriate structures.