rM. Thermoautotrophicus TDG with Buffer

TDG is a thermostable thymine DNA glycosylase from Methanobacterium thermoautotrophicum. The optimal temperature for the enzyme is 65°C. The enzyme lacks significant AP lyase or endonuclease activity. TDG works effectively in heteroduplex analysis to detect C to T transitions.

TDG enzyme recognizes T/G mismatches in duplex DNA and cleaves the strand with the T. The opposite strand is not cleaved. The enzyme also recognizes G/G mismatches if at least one nearest neighbor is an A or T and nicks one strand or the other. The enzyme exhibits poor AP lyase activity. One Unit is the amount of enzyme required to cleave 1 pmole of an oligonucleotide duplex containing a T/G mismatch in 1 hour at 65 °C. Only the strand containing the T is cleaved.

4 pmoles of T/G mismatch oligonucleotide set with the T oligo end-labeled, 1X REC Buffer 4 (10 mM HEPES-KOH (pH 7.4), 100 mM KCl, and 10 mM EDTA), and serial dilutions of enzyme in a 20 μL reaction volume are incubated for 1 hour at 65 °C. To complete cleavage of a basic site, fresh 1N NaOH is added to final concentration of 166 mM then heated for 15 minutes at 95 °C. For analysis, 24 μL of 2X Loading Buffer (20 mM EDTA, 95% formamide, and 0.13% bromophenol blue) are added, and the samples heated at 95 °C for 10 min then fast cooled to 2-8 °C. The cleavage products are resolved by 20% denaturing polyacrylamide gel electrophoresis, and percent cleavage quantified.