ThermoLabel Uracil DNA Glycosylase (ThermoLabel UDG) is a recombinant protein derived from psychrophilic marine bacteria expressed and purified in Escherichia coli, also known as Antarctic thermosensitive UDG. This thermosensitive UDG enzyme can effectively hydrolyze uracil on single or double stranded DNA, resulting in pyrimidine deficient sites that are easily hydrolyzed and broken at high temperatures or pH. This thermosensitive UDG enzyme has no activity against RNA and is mainly used for anti contamination of PCR amplification products. The uracil DNA glycosylase derived from Escherichia coli is relatively heat-resistant, and after 10 minutes of treatment at 95 ℃, a small amount of uracil DNA glycosylase activity remains, leading to the degradation of PCR products containing dU bases. The thermosensitive UDG derived from psychrophilic marine bacteria is completely inactivated at 50 ℃ for 5 minutes. Before PCR amplification, uracil DNA glycosylase (thermosensitive) is added to the PCR mixture, and residual contamination of the PCR product can be eliminated at 25 ℃ for 2 minutes. Due to the denaturation of uracil DNA glycosylase (thermosensitive) at 94 ℃ in the PCR cycle, it can be inactivated in one step, so it will not affect the new PCR product containing dU.
application
Removing uracil bases from single or double stranded DNA
Remove PCR residual contamination
Activity definition
Under the standard reaction system, the amount of enzyme required to catalyze the release of 60 pmol of uracil from double stranded DNA containing uracil per minute at 37 ° C is one active unit.
Reaction buffer
ThermoLabel Urecil DNA Glycosylase (UDG) is compatible with the vast majority of PCR polymerase reaction buffers, but its activity is inhibited at high ion concentrations (>100mM).
Enzyme preservation solution
20 mM Tris HCl, 50 mM NaCl, 1 mM DTT, 50% Glycerol, 0.1% (w/v) Triton X-100, pH 7.5.
Storage
-It can be stored for 2 years at 20 ℃.
Thermal inactivation
50 ℃ for 5 minutes.
Application examples in anti pollution PCR
To evaluate the ability of thermosensitive UDG to remove residual templates, we first amplified three gene products (GAPDH, EHP, Covid-19 N Gene) using RAPA3G Probe qPCR MasterMix containing dUTP. Then, 105 copies of U-containing products were used as templates and added to PCR final 5mU/µ l, 12.5mU/µ l, 25mU/µ l, and 50mU/µ l thermosensitive UDG (C5029) for qPCR. Incubate at 25 ℃ for 2 minutes to activate UDG, degrade U-containing products, and reduce its chances of generating false positive signals; Calculate the Ct values of three groups of heating sensitive UDG and non UDG reactions (as shown in the table below), and obtain △ Ct. The larger the △ Ct value, the higher the efficiency of removing residual products. This experiment demonstrates that a thermosensitive UDG of 5mU/µ l can effectively degrade U containing products (as shown in the figure below), indicating good ability to remove residual templates.
