Uracil-DNA Glycosylase, Heat-labile (Glycerol Free)
Uracil-DNA Glycosylase (UNG), Heat-labile
A heat-labile uracil-DNA glycosylase that hydrolyzes the N-glycosylic bond in uracil-containing DNA — used with dUTP to prevent carry-over contamination in PCR and RT-PCR. Also available in a glycerol-free version suited to lyophilization.
(≥40 U/μL)
Description
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 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.
Features
Prevents PCR carry-over
Useful in preventing PCR carry-over and false positives.
High thermal sensitivity
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.
Details
Storage condition
Components
| Product name | Conc. | Package | Code No. | Storage |
|---|---|---|---|---|
| Uracil-DNA Glycosylase, Heat-labile (Glycerol Free) | ≥40 U/μLL | 200 U×1 | TYB-UNG-201 | −20 °C |
Unit definition: 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 °C.
Application Data
Example 1. Confirmation of PCR product degradation capability
PCR were performed using dNTPs containing dUTP (Code No. TYB-NTP-501*), instead of dTTP. UNG was added to 100 ng of PCR product for degradation reaction, followed by electrophoresis. As a result, PCR products were confirmed to be degraded for 37 °C, 10 min. For inquiries about TYB-NTP-501, please contact support@diagnocine.com
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.
First PCR
Enteroviral RNA serves as template and amplified 196 bp targets by THUNDERBIRD® Probe One-step qRT-PCR Kit (Code No. TYB-QRZ-101). This kit contains dUTP.
Second PCR
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. TYB-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 2 U 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.
Ordering Information
Available versions under the Diagnocine catalog (TYB- prefix).
| Cat No. | Product | Conc. | Package | Order |
|---|---|---|---|---|
| TYB-UNG-101 | Uracil-DNA Glycosylase (UNG), Heat-labile | 1 U/µL | 200 U × 1 | Order |
| TYB-UNG-201 | Uracil-DNA Glycosylase (UNG), Heat-labile (Glycerol Free) | ≥40 U/µL | 200 U × 1 | Current page |
