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.

Roche Contact Information

Roche

Roche Diagnostics Corporation
P.O. Box 50414
9115 Hague Road
Indianapolis, IN 46250-0414 USA

Customer Service: 800-262-1640

Tech Support: 800-262-4911

Fax Number: 800-428-2883

Web Site: http://www.roche-applied-science.com

Uracil-DNA Glycosylase