Navigation Links
Preventing Carry-over Contamination with Uracil-DNA Glycosylase

The polymerase chain reaction (PCR) can amplify a single molecule over a billionfold. Thus, even minuscule amounts of a contaminant can be amplified and lead to a false positive result. Such contaminants are often products from previous PCR amplifications (carry-over contamination). Therefore, researchers have developed methods to avoid such contamination.

One common strategy is substituting dUTP for dTTP during PCR amplification, to produce uracil-containing DNA (U-DNA). Treating subsequent PCR reaction mixtures with Uracil-DNA Glycosylase (UNG) prior to PCR amplification and subsequent cleavage of apyriminic polynucleotides at elevated temperature (95C) under alkaline conditions (during the initial denaturation step) will remove contaminating U-DNA from the sample (see figure below). This method, of course, requires that all PCR-reactions in the lab have to be carried out with dUTP instead of dTTP.

Note the following when using dU-containing PCR products in downstream applications:
  • PCR products containing dU perform as well as those containing dT when used as hybridization targets or as templates for dideoxy sequencing.
  • PCR products containing dU can be cloned directly, if they are transformed into ungbacterial hosts.
  • A dU-containing substrate is readily digested by some common restriction enzymes (e.g. Eco RI and Bam HI), while others show reduced activity (e.g. Hpa I, Hind II, Hind III) on these substrates.
  • We do not recommend the use of dU -containing DNA for protein binding or DNA-protein interaction studies.

The following Roche Applied Science products are suited for preventing carry-over contamination in PCR:

Uracil-DNA Glycosylase (Cat. No. 1444646)
Uracil-DNA Glycosylase, heat-labile (Cat. No. 1775367)
PCR Core KitPLUS (Cat. No. 1585541)



Page: All 1 2

Related biology technology :

1. Combine High Yield, Great Accuracy and the Prevention of Carry-over Contamination by Using the Novel Expand High FidelityPLUS PCR System
2. Mycoplasma Contamination Reduces the Effect of Lipid-Mediated Transfection of Mammalian Cells
3. Combat RNase Contamination in the Lab
4. The Fastest, Simplest, and Most Effective Way to Remove DNA Contamination
5. Testing Compressed Air Lines for Microbiological Contamination
6. Testing Compressed Air Lines for Microbiological Contamination
7. Uracil-DNA Glycosylase
8. Uracil-DNA Glycosylase, heat-labile
Post Your Comments:

(Date:11/25/2015)... -- 2 nouvelles études permettent d , ... entre les souches bactériennes retrouvées dans la plaque ... . Ces recherches  ouvrent une nouvelle voie ... efficace de l,un des problèmes de santé les ...    --> 2 nouvelles études permettent d ...
(Date:11/25/2015)... HOLLISTON, Mass. , Nov. 25, 2015 ... HART ), a biotechnology company developing bioengineered organ implants ... McGorry will present at the LD Micro "Main ... 2:30 p.m. PT. The presentation will be webcast live ... Management will also be available at the conference for ...
(Date:11/25/2015)... 2015 /PRNewswire/ - Aeterna Zentaris Inc. (NASDAQ:  AEZS; TSX: ... prospects remain fundamentally strong and highlights the following ... received DSMB recommendation to continue the ZoptEC Phase ... the final interim efficacy and safety data ... men with heavily pretreated castration- and Taxane-resistant prostate ...
(Date:11/25/2015)... 25, 2015 Orexigen® Therapeutics, Inc. (Nasdaq: ... a fireside chat discussion at the Piper Jaffray 27th ... . The discussion is scheduled for Wednesday, December 2, ... .  A replay will be available for 14 days ... , Julie NormartVP, Corporate Communications and Business Development , ...
Breaking Biology Technology:
(Date:11/2/2015)... 2, 2015  SRI International has been awarded a ... development services to the National Cancer Institute (NCI) PREVENT ... scientific expertise, modern testing and support facilities, and analytical ... and toxicology studies to evaluate potential cancer prevention drugs. ... PREVENT Cancer Drug Development Program is an NCI-supported pipeline ...
(Date:10/29/2015)... , Oct. 29, 2015  Rubicon Genomics, ... for U.S. distribution of its DNA library preparation ... and Rubicon,s new ThruPLEX Plasma-seq kit. ThruPLEX Plasma-seq ... the preparation of NGS libraries for liquid biopsies--the ... diagnostic and prognostic applications in cancer and other ...
(Date:10/27/2015)... Oct. 27, 2015 In the present market ... concern for various industry verticals such as banking, healthcare, ... the growing demand for secure & simplified access control ... such as hacking of bank accounts, misuse of users, ... such as PC,s, laptops, and smartphones are expected to ...
Breaking Biology News(10 mins):