Non disponible en dehors du Royaume-Uni et de l'Irlande

Application

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.

Features and Benefits

Prevent carryover contamination in PCR. Increase the efficiency of site-directed mutagenesis procedures. Label oligonucleotide probes. Perform faster inactivation than the corresponding enzyme from E. coli due to the lower thermostability of the enzyme. Obtain no degradation of dU-PCR products within at least several hours of incubation at +2 to +8°C.ContentsThe enzyme is supplied as 1 U/µl solution in storage buffer.

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 CharacteristicsThe 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.

Legal Information

LightCycler is a registered trademark of Roche

Other Notes

For general laboratory use.

Quality

Function Test: Carryover prevention activity is assayed by adding approximately 105 dU that contains templates prior to the amplification reaction. After UNG treatment, no amplification products could be detected.The enzyme does not contain any contaminating exo- or endonucleases and is free from RNase activity, according to the current quality control procedures.

Specificity

Uracil-DNA glycosylase hydrolyzes uracil-glycosidic bonds at U-DNA sites in single- and doublestranded DNA, excising uracil and creating alkali sensitive abasic sites in the DNA. The enzyme is active on both single-stranded DNA and double-standed DNA. Uracil-DNA glycosylase is inactive on RNA and native, uracil-free DNA. Since uracil-DNA glycosylase has no metal ion requirements, it is fully active in the presence of EDTA.Heat inactivation: 95 °C for 2 min

Unit Definition

One unit is defined as the amount of Uracil-DNA Glycosylase required to completely degrade 1 µg purified single-stranded uracil-containing DNA (bacteriophage M13, grown in E. coli CJ236 dut-ung-) at +37 °C in 60 minutes.One Lindahl unit is defined as the amount of enzyme necessary to release 1 mol uracil at +37 °C in 1 minute. One Lindahl unit is comparable to 520 000 units based on our unit definition.Volume Activity: 1 U/µl