Uracil-DNA Glycosylase (UNG) hydrolyzes the N-glycosylic bond between the deoxyribose sugar and the base in uracil-containing DNA leaving an apyrimidinic site in DNA. Since DNA with an apyrimidinic site is degraded by heat treatment, UNG can be used in combination with dUTP to prevent carry-over of amplified products derived from PCR.
This enzyme hydrolyzes uracil from both single- and double-stranded DNA containing dU, but it not from RNA. It can also be used to prevent carryover of RT-PCR.
We also have a glycerol-free version that is suitable for lyophilization.
- Useful in preventing PCR carry-over and false positives
- Because of its high thermal sensitivity, it can be used not only for PCR but also for RT-PCR (RT-reaction should be performed at 42°C or higher)
|Product name||Conc.||Package||Code No.||Storage|
|Uracil-DNA Glycosylase (UNG),Heat-labile||1 U/μL||200 U×1||UNG-101||－20℃|
|Uracil-DNA Glycosylase (UNG),Heat-labile
|≧40 U/μL||200 U×1||UNG-201||－20℃|
One unit of UNG is defined as the amount of enzyme required to release 1 nmol uracil from uracil-containing DNA per hour at 37℃.
Example 1.Confirmation of PCR product degradation capability
PCR were performed using dNTPs containing dUTP (Code No. NTP-501), instead of dTTP. UNG was added to 100ng of PCR product for degradation reaction, followed by electrophoresis. As a result, PCR products were confirmed to be degraded for 37 ℃, 10min.
Example 2.Verification of the effect of preventing carry-over contamination
Assuming a case in which the amplified product splashed into the next PCR (carry-over contamination), a dilution of the amplified product was used as a template after UNG treatment. As a result, in the UNG treatment, the amplification product of the first PCR was degraded, and no amplification was observed in the second PCR.
Enteroviral RNA serves as template and amplified 196bp targets by THUNDERBIRD® Probe One-step qRT-PCR Kit(Code No. QRZ-101). This kit contains dUTP.
Amplified product on First PCR (104 copies) serves as template, the same target as the first PCR were amplified by THUNDERBIRD® Probe One-step qRT-PCR Kit (Code No. QRZ-101) supplemented with UNGs (0.4 U).
Example 3.Stability of PCR amplified products after UNG treatment.
If PCR is performed after adding UNG, the PCR product may be degraded before analysis when UNG is not completely deactivated. Our UNG was found to be completely inactivated during the PCR cycle, resulting in inactivation of the product and no degradation of the PCR amplification product.
Example 4.Effect of UNG-treatment on RT-PCR reaction
UNG 2U was added to the RT-PCR solution of THUNDERBIRD® Probe One-step qRT-PCR Kit(Code No. QRZ-101) and UNG-treated followed by real-time RT-PCR to detect enteroviral RNAs. As a result, there was a delay in the Ct value when the other companies’ heat-labile UNG was used, but there was no delay in the Ct value when our UNG was used.