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WarmStart® Afu Uracil-DNA Glycosylase (UDG)

Catalog # Concentration Size List Price Quantity Your Price
M1282S 2000 units/ml 200 units $138.00
$124.20
Catalog # Size List Price Your Price
M1282S 200 units $138.00
$124.20
Catalog #
Qty:
 
*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca
Mechanism diagram
  • Archaeoglobus fulgidus Uracil-DNA Glyocsylase
  • Thermostable monofunctional DNA glycosylase that catalyzes the hydrolysis of the N-glycosidic bond from deoxyuridine to release uracil
  • WarmStart technology inhibits enzyme activity below 42°C facilitating post isothermal amplification carryover prevention
  • Active on single-stranded and double-stranded DNA
  • Heat inactivation not possible
Formulated with a reversibly-bound aptamer that inhibits its activity at temperatures below 42°C, WarmStart Afu Uracil-DNA Glycosylase (UDG) from Archaeoglobus fulgidus efficiently catalyzes the release of free uracil from uracil-containing single-stranded or double-stranded DNA at temperatures >42°C.

Figure 1. WarmStart Afu Uracil-DNA Glycosylase (UDG) displays no detectable activity at temperatures below 42ºC.

Chart on Activity
(A) A temperature profile comparison of the UDG activity of Afu Uracil-DNA Glycosylase (UDG) (NEB #M0279) and WarmStart Afu Uracil-DNA Glycosylase (UDG) (NEB #M1282) illustrates the WarmStart inhibition of UDG activity below 42ºC. UDG activity was measured by incubating 0.002 U of either Afu Uracil-DNA Glycosylase (UDG) or WarmStart Afu Uracil-DNA Glycosylase (UDG) with 1.8 pmol of a fluorescently labeled 47-mer single-stranded DNA oligonucleotide containing a single uracil base in 30 minutes at various temperatures ranging from 25-65ºC. Following uracil excision, generated abasic sites were chemically cleaved (1M NaOH, 85°C for 10 minutes) before the reactions were analyzed by capillary electrophoresis (CE) fragment analysis on an Applied Biosystems 3730xl Genetic Analyzer (96 capillary array). The resulting activity demonstrates that while the WarmStart version of Afu Uracil-DNA Glycosylase (UDG) exhibits similar activity to Afu Uracil-DNA Glycosylase at 65°C, WarmStart Afu Uracil-DNA Glycosylase (UDG) has no detectable activity at 25°C.

(B) Strand Displacement Amplification (SDA) of a 500 bp amplicon of Lambda DNA was performed in the presence of dUTP at a low temperature range (30-45ºC) with WarmStart Afu Uracil-DNA Glycosylase (UDG) (top panel), with Afu Uracil-DNA Glycosylase (UDG) (middle panel) or without UDG (bottom panel). SDA generated a 500 bp band across the entire temperature range in the absence of UDG (bottom) but was completely inhibited by Afu Uracil-DNA Glycosylase (UDG) at temperatures >31ºC (middle). The inhibition of amplicon generation was due to the inherent activity of Afu Uracil-DNA Glycosylase (UDG) at low temperatures, which generates abasic sites in dU-containing amplicons, thus preventing them from being further amplified. The presence of WarmStart Afu Uracil-DNA Glycosylase (UDG) (top), however, has almost no effect on the amplification of dU-containing amplicons, except a slight decrease of product yield around 45°C, indicating that WarmStart Afu Uracil-DNA Glycosylase (UDG) is active at temperatures >42°C.



Figure 2. WarmStart Afu Uracil-DNA Glycosylase (UDG) efficiently digests uracil-containing amplification products at 65ºC.

Graph of Digestion
 
(A) Schematic illustration of post-SDA amplification carryover prevention with WarmStart Afu Uracil-DNA Glycosylase (UDG). A 500 bp amplicon was amplified in the presence of dUTP containing no UDG or WarmStart Afu Uracil-DNA Glycosylase (UDG) at 34ºC, where WarmStart Afu Uracil-DNA Glycosylase (UDG) is inactive. Following SDA, the reactions were incubated at 65ºC for 20 minutes to generate abasic sites at the locations of previously incorporated dUs. The DNA backbone at abasic sites was then cleaved by incubating at 85ºC for 10 minutes under alkaline conditions.

(B) After amplification and treatment at 65ºC for 20 minutes, both the No UDG and WarmStart Afu Uracil-DNA Glycosylase (UDG) samples had similar yields of the 500 bp amplicon as visualized by gel electrophoresis. The DNA backbone at abasic sites was then cleaved by incubating at 85ºC for 10 minutes under alkaline conditions. After chemical cleavage, the 500 bp products remained intact in reactions without UDG but became much weaker bands and smeared products in reactions containing WarmStart Afu Uracil-DNA Glycosylase (UDG).



Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  WarmStart® Afu Uracil-DNA Glycosylase (UDG) M1282SVIAL -20 1 x 0.1 ml 2,000 units/ml
  ThermoPol® II (Mg-free) Reaction Buffer B9005SVIAL -20 1 x 1.5 ml 10 X
An exception occurred during the operation, making the result invalid. Check InnerException for exception details.

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