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.

Fig. 1

Preparation of the heteroduplex EGFP plasmid. (A) Flow chart for the preparation of heteroduplex EGFP plasmid. pGEM5Z(+)-EGFP (also named p111) was nicked with Nb.Bpu10I in a 400 μl reaction volume containing 20 μg pGEM5Z(+)-EGFP, 4 μl (20 u) Nb.Bpu10I, 40 μl 10× Buffer and nuclease-free water at 37°C for 1 hour. After phenol/chloroform extraction and ethanol precipitation, the nicked plasmid was digested with 1200 u Exonuclease III for about 10 minutes at 30°C to degrade the nicked strand. p189 was linearized with restriction enzyme AseI and mixed with the purified circular ssDNA at a ratio of 1:1.5 (heat at 90°C for 3 minute and cool down to room temperature by leaving the heater block on the bench) to generate a heteroduplex EGFP plasmid containing a G/T mismatch and a nick. The annealing mixture was treated with the plasmid-safe ATP-dependent DNase (PSAD) to degrade leftover ssDNA and linearized plasmid DNA. The heteroduplex EGFP plasmid with high purity was recovered using a DNA clean up kit. (B) Comparison of the ssDNA generated with the enzyme digestion approach described above in A (Lane1), and with a phagemid pGEM5Z(+)-EGFP (4.6kb) using our previously described approach in [8] (Lane2). (C) Electrophoretic analysis of various DNA species: pGEM5Z(+)-EGFP isolated with an alkaline lysis method containing supercoiled (SC), relaxed (R), and nicked circular (N) DNA (Lane 1), circular ssDNA(Lane 2), linear form of p189 (4.7 kb) (Lane 3), purified heteroduplex EGFP plasmid with a nick (Lane 4), and DNA ladder (Lane 5).

Fig. 1

Preparation of the heteroduplex EGFP plasmid. (A) Flow chart for the preparation of heteroduplex EGFP plasmid. pGEM5Z(+)-EGFP (also named p111) was nicked with Nb.Bpu10I in a 400 μl reaction volume containing 20 μg pGEM5Z(+)-EGFP, 4 μl (20 u) Nb.Bpu10I, 40 μl 10× Buffer and nuclease-free water at 37°C for 1 hour. After phenol/chloroform extraction and ethanol precipitation, the nicked plasmid was digested with 1200 u Exonuclease III for about 10 minutes at 30°C to degrade the nicked strand. p189 was linearized with restriction enzyme AseI and mixed with the purified circular ssDNA at a ratio of 1:1.5 (heat at 90°C for 3 minute and cool down to room temperature by leaving the heater block on the bench) to generate a heteroduplex EGFP plasmid containing a G/T mismatch and a nick. The annealing mixture was treated with the plasmid-safe ATP-dependent DNase (PSAD) to degrade leftover ssDNA and linearized plasmid DNA. The heteroduplex EGFP plasmid with high purity was recovered using a DNA clean up kit. (B) Comparison of the ssDNA generated with the enzyme digestion approach described above in A (Lane1), and with a phagemid pGEM5Z(+)-EGFP (4.6kb) using our previously described approach in [8] (Lane2). (C) Electrophoretic analysis of various DNA species: pGEM5Z(+)-EGFP isolated with an alkaline lysis method containing supercoiled (SC), relaxed (R), and nicked circular (N) DNA (Lane 1), circular ssDNA(Lane 2), linear form of p189 (4.7 kb) (Lane 3), purified heteroduplex EGFP plasmid with a nick (Lane 4), and DNA ladder (Lane 5).

Fig. 2

Repair of heteroduplex EGFP plasmid in live cells. HeLa (MMR-proficient, A, B) and HCT116 cells (MMR-deficient, C, D) co-transfected with the heteroduplex EGFP plasmid and pDsRed1-N1. Flow cytometry results are shown on the right side.