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:10/12/2017)... ... October 12, 2017 , ... ... multicenter, prospective clinical study that demonstrates the accuracy of the FebriDx® test, ... clinically significant acute bacterial and viral respiratory tract infections by testing the ...
(Date:10/12/2017)... ... October 12, 2017 , ... ... the first-ever genomics analysis platform specifically designed for life science researchers to ... of pioneering researcher Rosalind Franklin, who made a major contribution to the ...
(Date:10/11/2017)... NJ (PRWEB) , ... October 11, 2017 , ... Personal eye wash is a basic ... one eye at a time. So which eye do you rinse first if a dangerous ... have Plum Duo Eye Wash with its unique dual eye piece. , “Whether ...
(Date:10/11/2017)... , Oct. 11, 2017  VMS BioMarketing, a leading provider ... nationwide oncology Clinical Nurse Educator (CNE) network, which will launch ... for communication among health care professionals to enhance the patient ... office staff, and other health care professionals to help women ... cancer. ...
Breaking Biology Technology:
(Date:6/23/2017)... ITHACA, N.Y. , June 23, 2017 /PRNewswire/ ... leader in dairy research, today announced a new collaboration ... reduce the chances that the global milk supply is ... dairy project, Cornell University has become the newest academic ... Supply Chain, a food safety initiative that includes IBM ...
(Date:5/6/2017)... 5, 2017 RAM Group , ... new breakthrough in biometric authentication based on a ... properties to perform biometric authentication. These new sensors are ... created by Ram Group and its partners. This sensor ... supply chains and security. Ram Group is a ...
(Date:4/13/2017)... Calif. , April 13, 2017 UBM,s ... York will feature emerging and evolving technology ... Both Innovation Summits will run alongside the expo portion ... speaker sessions, panels and demonstrations focused on trending topics ... largest advanced design and manufacturing event will take place ...
Breaking Biology News(10 mins):