Cold UDG (Uracil-DNA Glycosylase) is quickly and irreversibly inactivated at mild heating condition (~50℃). The enzyme is engineered through super-computer assisted screening method. The hub amino acid residues important for maintaining structural stability of UDG were predicted by Laplacian clustering analysis. Iterative site-directed mutagenesis followed by expression and purification of recombinant engineered UDG were conducted. Finally, we select the most thermolabile UDG with the highest specific activity; Cold UDG. The enzyme can be applied for the prevention of carryover contamination in qPCR, and also in RT-qPCR.
- Heat inactivated at temperatures above 50°C
- Molecular weight: 24.5 kDa
- Reaction temperature: 37℃
- Control of carry-over contamination in PCR
- Cloning of PCR products
- Used to investigate features of protein-DNA interactions
- Purity: >99% on SDS-PAGE
One unit is defined as the amount of enzyme that catalyzes the release of 60 pmol of uracil per minute from double-stranded, uracil containing DNA
10 mM Tris-HCl, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.1 mg/ml BSA, 50% Glycerol pH 7.4
Figure 1. Thermal inactivation of Cold UDG
Residual activity of Cold UDG and E.coli UDG after heat treatment at different temperature of 35~95°C. Cold UDG show great thermolability compare to E.coli UDG
Figure 2. Prevent PCR carryover contamination
qPCR assay was performed using serial diluted dU-containing PCR amplified DNA as a template. Cold UDG effectively removed dU-containing contaminated DNA and delayed cycle threshold from 7~9 (Red: w/o Cold UDG, Others: w/ cold UDG).