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Published on: April 5, 2018
False negative results from using common PCR reagents
Dean J Bacich1, Kathryn M Sobek, Jessica L Cummings
1Department of Urology, University of Pittsburgh, 5200 Centre Avenue, Pittsburgh, PA 15232, USA. okeefeds@upmc.edu.
BMC Research Notes
|October 29, 2011
Summary
Uracil-DNA-glycosylase (UNG) prevents PCR contamination but can cause false negatives. Minute amounts of UNG-treated PCR products or primer-dimers can inhibit amplification of legitimate DNA targets.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Polymerase Chain Reaction (PCR) is sensitive for detecting novel viruses.
- Carry-over contamination is a known issue in PCR.
- Uracil-DNA-glycosylase (UNG) is commonly used in PCR master mixes to prevent contamination.
Purpose of the Study:
- To investigate the impact of UNG on PCR sensitivity.
- To identify potential causes of false-negative PCR results.
Main Methods:
- Testing PCR assay sensitivity for murine DNA contamination.
- Evaluating the effect of UNG-digested PCR products and primer-dimers on amplification.
Main Results:
- Minute quantities of UNG-digested PCR product can inhibit amplification.
- Primer-dimers in negative controls can also block legitimate DNA amplification.
- UNG presence or absence did not alter the inhibitory effect of these contaminants.
Conclusions:
- UNG, while preventing contamination, may lead to false-negative PCR results.
- This phenomenon could explain discrepant findings in studies of MLV-related viruses.
- Careful control of potential false negatives is crucial in low-target-copy-number PCR experiments.
