Uses
Uracil-DNA glycosylase can be used to cleave DNA at any site where a deoxyuridylate residue has been incorporated. Because of the heat-lability of Uracil-DNA Glycosylase, heat labile the main application of this enzyme is the prevention of carryover contamination in PCR. In contrast to the enzyme from E.coli; using this preparation of UNG it is not necessary to freeze the PCR product immediately after amplification or to hold the reaction mixture at +70 °C.
Important Note: For highly sensitive techniques like real-time PCR we recommend our LightCycler? Uracil-DNA Glycosylase which is optimized for this application.
General Description
Uracil-DNA Glycosylase, heat-labile contains the equally named enzyme found in the marine bacterium BMTU 3346. Like the UNG from E. coli it hydrolyzes uracil-glycosidic bonds in single- or double-stranded DNA, excising uracil and creating alkali-sensitive abasic sites in the DNA. These abasic sites can be hydrolyzed by endonuclease, heat, or alkali treatment. Depending on how the DNA is prepared, Uracil-DNA Glycosylase can be used to achieve general, site-specific, or strand-specific U-DNA cleavage. This enzyme shows lower thermostability and is therefore easier to inactivate.
Enzyme Characteristics
The BMTU 3346 enzyme is inactivated more quickly (2 minutes at +95°C) than the corresponding enzyme from E. coli (10 minutes at +95°C). It has also been reported that UNG from E. coli remains partially active, leading to the degradation of the dU-containing PCR product. In contrast, the heat-labile UNG does not degrade dU-PCR products within at least several hours of incubation at +2 to +8°C. Therefore, it is not necessary to freeze the PCR product immediately after amplification or to hold the reaction mixture at -70°C.
