Thomas Evangelos, Pingoud Alfred, Friedhoff Peter
Institut für Biochemie, Justus-Liebig-Universität, Giessen, Germany.
Biol Chem. 2002 Sep;383(9):1459-62. doi: 10.1515/BC.2002.166.
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.
我们提出了一种方法，该方法无需使用凝胶电泳或色谱纯化步骤即可制备含有特定错配的长DNA。制备过程始于合成两个PCR产物，除了那些随后将形成错配的位置外，这两个产物是相同的。其中一个PCR引物必须进行5'-磷酸化，这样在两个单独的反应中可获得两个PCR产物，其中一条链进行了5'-磷酸化。用λ-外切核酸酶去除磷酸化链后，将所得单链杂交形成含错配的异源双链体。该方法在大肠杆菌MutHLS系统分析中的应用得到了证明。
