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.
(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.
(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).
This product is related to the following categories:
The following reagents are supplied with this product:
NEB #
Component Name
Component #
Stored at (°C)
Amount
Concentration
M1282S
-20
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
Properties & Usage
Unit Definition
One unit is defined as the amount of enzyme required to release 60 pmol per minute of a fluorescently labeled 47 mer single-stranded DNA oligonucleotide containing a single uracil base in 30 minutes at 65°C in a total reaction volume of 50 µl in 1X Thermopol II Buffer.
Reaction Conditions
1X ThermoPol® II (Mg-free) Reaction Buffer
Incubate at 65°C
1X ThermoPol® II (Mg-free) Reaction Buffer
20 mM Tris-HCl
10 mM (NH4)2SO4
10 mM KCl
0.1% Triton® X-100
(pH 8.8 @ 25°C)
Storage Buffer
10 mM Tris-HCl
50 mM KCl
1 mM DTT
0.1 mM EDTA
0.1 mg/ml Recombinant Albumin
50% Glycerol
pH 7.4 @ 25°C
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