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E. Thomas, A. Pingoud, P. Friedhoff

An Efficient Method for the Preparation of Long Heteroduplex DNA as Substrate for Mismatch Repair by the Escherichia coli MutHLS System

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 5phosphorylated, such that in two separate reactions two PCR products are obtained, which are 5phosphorylated in one strand. After removal of the phosphorylated strands by ?exonuclease, the resulting single strands are hybridized to form the mismatchcontaining heteroduplex. The application of this procedure is demonstrated for the analysis of the Escherichia coli MutHLS system.

Biological Chemistry, Walter de Gruyter

Print ISSN: 1431-6730
Volume: 383, 09/2002
Pages: 1459 - 1462

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