What does UNG enzyme do?

Uracil-DNA glycosylase is also known as UNG or UDG. Its most important function is to prevent mutagenesis by eliminating uracil from DNA molecules by cleaving the N-glycosidic bond and initiating the base-excision repair (BER) pathway.

What is AmpErase?

AmpErase UNG (Uracil N-glycosylase) is an enzyme utilized in a powerful method for elimination of carryover PCR products in Real-Time PCR.

What is the function of Uracil-DNA glycosylase in PCR?

Uracil-DNA glycosylase (UNG) is a DNA repair enzyme that will cleave uracil-containing DNA while leaving the natural, thymine-containing DNA unaffected [7, 8]. During PCR, deoxyuridine triphosphate (dUTP) can be substituted for deoxythymidine triphosphate (dTTP) in the synthesis of product DNA.

How does UDG work?

UDG cleaves the uracil base from the phosphodiester backbone of uracil-containing DNA, but has no effect on natural (i.e., thymine-containing) DNA. The resulting apyrimidinic sites block replication by DNA polymerases, and are very labile to acid/base hydrolysis.

What is UNG activation?

UNG allows previous PCR amplifications or mis-primed, nonspecific products to degrade, leaving native nucleic acid templates intended for amplification intact. UNG activation occurs as the first step of PCR at a 50°C incubation for 2 minutes.

What is UNG incubation for PCR?

2 minutes
UNG activation occurs as the first step of PCR at a 50°C incubation for 2 minutes.

What is the advantage of using dUTP in high throughput PCR analysis?

Prevent Carryover from Previous PCR Amplifications Promega dNTPs have greater than 99% triphosphate content and are provided at a concentration of 100mM in water at pH 7.5. dUTP (2´-Deoxyuridine, 5´-Triphosphate) can be used in place of dTTP in PCR and RT-PCR protocols to prevent carryover from previous amplifications.

What is the function of DNA glycosylase?

DNA glycosylases play a key role in the elimination of such DNA lesions; they recognize and excise damaged bases, thereby initiating a repair process that restores the regular DNA structure with high accuracy.

What is UNG in qPCR?

Family I UDG enzymes are called UNG, after the uracil-N-glycosylase gene [6]. The terms UDG and UNG are commonly used interchangeably because they perform the same function in qPCR—namely to prevent carryover contamination.

Is dUTP used in PCR?

In PCR, deoxythymidine triphosphate (dTTP) can be replaced with deoxyuridine triphosphate (dUTP). Before PCR is initiated, the use of uracil-DNA N-glycosylase (UNG) will degrade any uracil-containing PCR products, i.e., eliminate carry-over contamination [19].

How does PCR prevent water evaporation?

Ensure that your tubes are tightly closed prior to PCR. As a last resort, you could try adding 30 uL mineral oil to the top of the reaction mix to prevent any kind of evaporation.

What is function of DNA glycosylase involved in base excision repair?

Enzymes that cleave the bond between deoxyribose and a modified or mismatched DNA base are now called DNA glycosylases. Collectively these enzymes initiate base excision repair of a large number of base lesions, each recognized by one or a few DNA glycosylases with overlapping specificities.

What is a glycosylase enzyme?

DNA glycosylases are a family of enzymes involved in base excision repair, classified under EC number EC 3.2. 2. Base excision repair is the mechanism by which damaged bases in DNA are removed and replaced. DNA glycosylases catalyze the first step of this process.

How does a Glycosylase work?

DNA glycosylases recognize and remove damaged bases from DNA by cleaving the base–sugar (N-glycosylic) bond, and downstream base excision repair enzymes restore the correct nucleotide.

What is dUTP in DNA?

dUTP is a close structural congener of dTTP and can be readily incorporated into DNA opposite to adenine during DNA replication leading to non-mutagenic dU/A base pairs (‘uracilation’).