Active in Fermentas buffers for restriction enzymes and thermophilic polymerases.
Applications
Control of carry-over contamination in PCR (2).
Glycosylase mediated single nucleotide polymorphism detection (GMPD) (3).
Site-directed mutagenesis (4).
As a probe for protein-DNA interaction studies (5).
SNP genotyping.
Cloning of PCR products (6).
Generation of single strand overhangs of PCR products and cDNA.
Description
Uracil-DNA Glycosylase (UDG, UNG) catalyzes the hydrolysis of the N-glycosylic bond between uracil and sugar, leaving an apyrimidinic site in uracil-containing single or double-stranded DNA, see Fig.1. Shows no activity on RNA (1).
Source
E.coli K12 cells.
Molecular Weight
25.6 kDa monomer.
Definition of Activity Unit
One unit of the enzyme catalyzes the release 1 nanomole of uracil from uracil-containing DNA template in 60 min at 37°C.
Storage Buffer
The enzyme is supplied in: 30 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.05% (v/v) Tween 20 and 50% (v/v) glycerol.
10X Reaction Buffer
200 mM Tris-HCl (pH 8.2 at 25°C), 10 mM EDTA, 100 mM NaCl.
Quality Control
The absence of endo-, exodeoxyribonucleases, phosphatases and ribonucleases confirmed by appropriate quality tests.
Inhibition and Inactivation
Inhibitors: Ugi protein from the Bacillus subtilis phage PBS2, protein p56 from the Bacillus subtilis phage phi29 (7).
Inactivated by heating at 95°C for 10 min. Enzyme activity is partially restored at temperatures lower than 55°C. Therefore put PCR products on ice after PCR and load directly on a gel.
Note
The abasic sites formed in DNA by Uracil-DNA Glycosylase may be subsequently cleaved by heat, alkali-treatment or endonucleases that cleave specifically at abasic sites.
UDG (UNG) is active in the presence or absence of divalent cations.
Notice
Use of this enzyme in certain applications may be covered by patents and may require a license.
Lindahl, T., et al., DNA N-Glycosidases, J. Biol. Chem., 252, 10, 3286-3294, 1977.
Longo, M.C., et al., Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions, Gene, 93, 125-128, 1990.
Vanghan, P., McCarthy, T.V., A novel process for mutation detection using uracil DNA glycosylase, Nucleic Acids Res., 26, 810-815, 1998.
Kunkel, T.A., Rapid and efficient site-specific mutagenesis without phenotypic selection, Proc. Natl. Acad. Sci. USA, 82, 488-492, 1985.
Devchand, P.R., et al., Uracil-DNA glycosylase as a probe for protein-DNA interactions, Nucleic Acids Res, 21, 3437-3443, 1993.
Booth, P.M., et al., Assembly and cloning of coding sequences for neurotrophic factors directly from genomic DNA using polymerase chain reaction and uracil DNA glycosylase, Gene, 146, 303-308, 1994.
Gamma Serrano-Heras et al., Protein p56 from the Bacillus subtilis phage phi29 inhibits DNA-binding ability of uracil-DNA glycosylase, Nucleic Acids Res, 13, 1-9, 2007.
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- to print
- Tango™ buffer for 100% activity
- B buffer for 100% activity
- G buffer for 100% activity
- Thermal inactivation at 95°C in 10 min
- LO certified
Figure 1. Uracil-DNA Glycosylase activity.
