Product Class: Other

WarmStart® Afu Uracil-DNA Glycosylase (UDG)
Gray property icon for NEBU Gray property icon for cloned at NEB recombinant 65 Gray property icon for heat inactivation no Gray property icon for Hot Start

Product Introduction

Mechanism of WarmStart Afu UDG

  • 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
Bioz Badge Exists : True
Catalog # Size Concentration
M1282S 200 units 2000 units/ml

Product Information

Description

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

Line graph and gel showing inhibition of UDG below 42°C for WarmStart Afu UDG
(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 

 Workflow showing mechanism of WarmStart Afu UDG, followed by gel showing efficient digestion of UDG
 
(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:
DNA Repair Enzymes and Structure-specific Endonucleases,

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • NEB # Stored at (°C)
    M1282S -20
    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

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

Heat Inactivation

No

Molecular Weight

Theoretical: 22717 daltons

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  1. What is the activity of WarmStart Afu Uracil-DNA Glycosylase (UDG) in NEBuffers?
  2. Will WarmStart Afu Uracil-DNA Glycosylase (UDG) work in Thermopol® Buffer?
  3. What is the molecular weight of WarmStart Afu Uracil-DNA Glycosylase (UDG)?
  4. Is WarmStart Afu Uracil-DNA Glycosylase (UDG) a tagged protein?
  5. Does the Uracil Glycosylase inhibitor (UGI) inhibit WarmStart Afu Uracil-DNA Glycosylase (UDG)?
  6. Does WarmStart Afu Uracil-DNA Glycosylase (UDG) release uracil from ss and dsDNA?
  7. Does WarmStart Afu Uracil-DNA Glycosylase (UDG) cut RNA?
  8. Will WarmStart Afu Uracil-DNA Glycosylase (UDG) work in Fpg buffer?
  9. What does WarmStart mean?
  10. How can WarmStart Afu Uracil-DNA Glycosylase (UDG) be used for carryover prevention?
  11. What is Strand Displacement Amplification (SDA)?

Quality, Safety & Legal

Quality Assurance Statement

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Specifications

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Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

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