Preparation of heteroduplex enhanced green fluorescent protein plasmid for in vivo mismatch repair activity assay

Preparation of heteroduplexes in large quantities with high purity is essential for the measurement of DNA mismatch repair (MMR) activity. Here we report a rapid, less labor-intensive method for the preparation of a heteroduplex plasmid that expresses the enhanced green fluorescent protein (EGFP) if the mismatch is repaired correctly. The method involves the use of a wild-type and a mutated EGFP expression plasmid and a few steps of enzymatic digestion. When the constructed heteroduplex EGFP plasmid was transfected into MMR-proficient and -deficient cell lines, the number of EGFP-expressing cells was Much higher in the MMR-proficient cells than in the MMR-deficient cells, suggesting that the heteroduplex can be used for MMR activity assay in live model systems. (C) 2009 Elsevier Inc. All rights reserved.