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.
Zhou
Bisheng
B
Institute of Watershed Science and Environmental Ecology, Wenzhou Medical College, 325035 Wenzhou, People's Republic of China.
Huang
Changjiang
C
Yang
Junhua
J
Lu
Jianxin
J
Dong
Qiaoxiang
Q
Sun
Lu-Zhe
LZ
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
D009692:Nucleic Acid Heteroduplexes; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004247:DNA