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)


'"/>

Source:


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:
*Name:
*Comment:
*Email:


(Date:2/16/2017)... WEST PALM BEACH, Fla. , Feb. 16, ... molecular diagnostics company revolutionizing the development of liquid ... that it has entered into an exclusive license ... who will distribute MDNA,s proprietary liquid biopsy test ... in South Korea . This ...
(Date:2/16/2017)... 16, 2017   Biostage, Inc. (Nasdaq: ... company developing bioengineered organ implants to treat cancers and ... announced today the closing on February 15, 2017 of ... common stock and warrants to purchase 20,000,000 shares of ... The offering was priced at $0.40 per share of ...
(Date:2/16/2017)... , Feb. 16, 2017  Champions Oncology, Inc. ... the development and sale of advanced technology solutions and ... drugs, today announced the addition of new cohorts of ... These new models will expand Champions, product line in ... and neck cancer, AML, and non-small cell lung cancer ...
(Date:2/16/2017)... and NEW YORK , Feb. 16, ... completion of their $7M Series B financing, adding an ... the $3.5M led by Mesa Verde Venture Partners and ... resources will be directed towards further accelerating commercial adoption ... comprehensive genomic profiling test and expanding the Paradigm cancer ...
Breaking Biology Technology:
(Date:2/10/2017)... 10, 2017 Research and Markets ... "Personalized Medicine - Scientific and Commercial Aspects" to their ... ... is integrated with therapy for selection of treatment as well ... and prevention of disease in modern medicine. Biochip/microarray technologies and ...
(Date:2/8/2017)... 2017  Aware, Inc. (NASDAQ: AWRE ), a ... results for its quarter and year ended December 31, 2016. ... was $3.9 million compared to $6.9 million in the same ... 2016 was $0.6 million compared to $2.6 million in the ... of 2016 was $0.5 million, or $0.02 per diluted share, ...
(Date:2/8/2017)... Feb. 7, 2017 Report Highlights ... 2021 from $8.3 billion in 2016 at a compound ... 2021. Report Includes - An overview of the ... trends, with data from 2015 and 2016, and projections ... Segmentation of the market on the basis of product ...
Breaking Biology News(10 mins):