Figure 4.

Cleavage of A/C mismatch in K-ras G13D sequence amplified from colon cancer cell lines by Tma endo V mutant Y80A. (A) Schematic illustration of blunt end and sticky end heteroduplex G12V and G13D PCR products. See Materials and Methods for details. (B) Cleavage of G13D by Tma endo V mutant Y80A. Cleavage reaction mixtures (10 μl) containing 10 mM HEPES-KOH (pH 7.4), 1 mM DTT, 2% glycerol, 2.5 mM MnCl₂, 100 ng of wild-type K-ras homoduplex or G12V heteroduplex or G13D heteroduplex and 100 nM Tma endo V mutant Y80A protein were incubated at 65°C for 30 min. For the reactions that were followed by ligation, the amount of K-ras homoduplex or heteroduplex was increased to 200 ng in the cleavage reactions. The cleavage reaction mixtures were filtered through an YM-10 microcon spin column and washed with TE buffer containing 10 mM Tris–HCl (pH 7.6) and 1 mM EDTA. To seal the non-specific nicks, the washed cleavage reaction mixtures (in 6 μl TE) were supplemented with 1 μl of 10 × Taklig buffer [20 mM Tris–HCl (pH 7.6), 100 mM KCl, 10 mM DTT, 20 μg/ml BSA], 1 μl of 100 mM MgCl₂, 1 μl of 10 mM NAD⁺ and 1 μl of 20 nM Tsp AK16D ligase. The ligation mixtures were incubated at 65°C for 20 min.