title: Preparation of heteroduplex enhanced green fluorescent protein plasmid for in vivo mismatch repair activity assay.
abstract: 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.
authors.affiliation: Institute of Watershed Science and Environmental Ecology, Wenzhou Medical College, 325035 Wenzhou, People's Republic of China.
authors.forename: Bisheng
authors.initials: B
authors.lastname: Zhou
authors.forename: Changjiang
authors.initials: C
authors.lastname: Huang
authors.forename: Junhua
authors.initials: J
authors.lastname: Yang
authors.forename: Jianxin
authors.initials: J
authors.lastname: Lu
authors.forename: Qiaoxiang
authors.initials: Q
authors.lastname: Dong
authors.forename: Lu-Zhe
authors.initials: LZ
authors.lastname: Sun
mesh_terms: D004247:DNA / Q000378:metabolism*; D053843:DNA Mismatch Repair; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism*; D010957:Plasmids
chemical_list: D009692:Nucleic Acid Heteroduplexes; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004247:DNA