An efficient method for the preparation of long heteroduplex DNA as substrate for mismatch repair by the Escherichia coli MutHLS system
An efficient method for the preparation of long heteroduplex DNA as substrate for mismatch repair by the Escherichia coli MutHLS system
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DOI：
10.1515/bc.2002.166
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发表时间：
2002-09-01
影响因子：
3.7
通讯作者：
Friedhoff, P
中科院分区：
文献类型：
--
作者：
Thomas, E;Pingoud, A;Friedhoff, P
我们提出了一种方法，允许制备含有定义的错配的长DNA，而不使用凝胶电泳或色谱纯化步骤。制备从合成两个PCR产物开始，除了稍后将形成错配的那些位置之外，这两个PCR产物是相同的。PCR引物之一必须是5 '-磷酸化的，使得在两个单独的反应中获得两个PCR产物，其在一条链中是5'-磷酸化的。在通过外切核酸酶去除磷酸化链后，所得单链杂交以形成含有错配的异源双链体。该程序的应用证明了大肠杆菌MutHLS系统的分析。
We present a method that allows preparing long DNA containing defined mismatches without the use of gel electrophoretic or chromatographic purification steps. The preparation starts with the synthesis of two PCR products, which are identical except for those positions that will later form the mismatches. One of the PCR primers must be 5'-phosphorylated, such that in two separate reactions two PCR products are obtained, which are 5'-phosphorylated in one strand. After removal of the phosphorylated strands by lambda-exonuclease, the resulting single strands are hybridized to form the mismatch-containing heteroduplex. The application of this procedure is demonstrated for the analysis of the Escherichia coli MutHLS system.