Uracil-DNA Glycosylase

for prevention of carry-over contamination in PCR Cat. No. 1 444 646 100 units
Cat. No. 1 269 062 25 units

Description

Uracil-DNA Glycosylase (UNG) (from E. coli) catalyzes the removal of uracil from single- and double-stranded DNA that has been synthesized in the presence of dUTP. The apyriminic sites formed by UNG are susceptible to cleavage by heat under alkaline conditions (carry-over prevention). UNG will not digest 3'-terminal dU, dUTP, primers labeled with biotin-dUTP, or digoxigenin-dUTP. Ribouracil residues in RNA are also unaffected by UNG.

Application

Prevention of carry-over contamination in:

PCR with Taq DNA Polymerase.
RT-PCR with C. therm. Polymerase One-step RT-PCR System.

Operating parameters

Substrate: Single- or double-stranded DNA containing deoxyuracil (DNA synthesized in the presence of dUTP).
Incubation conditions for cleavage of uracil-glycosidic bonds: 20°C, 1–10 min.
Thermal stability (pH 8.3): half-life at 40°C, 2 min; half-life at 45°C, 0.5 min.
MgCl₂ concentration: 2.5 mM.
dNTP concentration: 200 µM dNTPs; 600 µM dUTP.
Conditions for inactivation of enzyme: 95°C, 2 min.

Notes:
UNG (2 units) will degrade up to 10⁶ uracil-containing DNA molecules in10 min. In contrast to the enzyme from E. coli, heat-labile UNG will not modify dU-containing PCR products for at least several hours at 4°C. Therefore, it is not necessary to freeze dU-containing PCR products immediately after amplification, or to hold the reaction mixture at 72°C until it is used. However, note that after prolonged storage (>8 h) residual enzyme activity may lead to degradation of PCR product. For long term storage, PCR product should therefore be frozen at –20°C.

Key advantages

Prevents carry-over contamination in PCR, because the enzyme modifies all uracil-containing products from previous PCRs. These products are then hydrolyzed by heat under alkaline conditions.
Does not degrade native (thymidine-containing) templates, because the enzyme is free of contaminating exo- and endonucleases.
Does not interfere with PCR amplification, because the enzyme is not active at temperatures typically used for PCR applications.