An efficient method for the preparation of long heteroduplex DNA as substrate for mismatch repair by the Escherichia coli MutHLS system.

Abstract:

: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.

journal_name

Biol Chem

journal_title

Biological chemistry

authors

Thomas E,Pingoud A,Friedhoff P

doi

10.1515/BC.2002.166

keywords:

subject

Has Abstract

pub_date

2002-09-01 00:00:00

pages

1459-62

issue

9

eissn

1431-6730

issn

1437-4315

journal_volume

383

pub_type

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