proven in actual studies.
False negative results from using common PCR reagents
The sensitivity of the PCR reaction makes it ideal for use when
identifying potentially novel viral infections in human disease.
Unfortunately, this same sensitivity also leaves this popular
technique open to potential contamination with previously amplified
PCR products, or "carry-over"contamination.
PCR product carry-over contamination can be prevented with
uracil-DNA-glycosylase (UNG), and it is for this reason that it is
commonly included in many commercial PCR master-mixes. While testing
the sensitivity of PCR assays to detect murine DNA contamination in
human tissue samples, we inadvertently discovered that the use of this
common PCR reagent may lead to the production of false-negative PCR
results.
Findings: We show here that contamination with minute quantities of
UNG-digested PCR product or any negative control PCR reactions
containing primer-dimers regardless of UNG presence can completely
block amplification from as much as 60ng of legitimate target DNA.
Conclusions: These findings could potentially explain discrepant
results from laboratories attempting to amplify MLV-related viruses
including XMRV from human samples, as none of the published reports
used internal-tube controls for amplification.
The potential for false negative results needs to be considered and
carefully controlled in PCR experiments, especially when the target
copy number may be low - just as the potential for false positive
results already is.
Author: Dean Bacich, Kathryn Sobek, Jessica Cummings, Allison Atwood,
Denise O'Keefe
Credits/Source: BMC Research Notes 2011, 4:457
Published on: 2011-10-27
http://www.biomedcentral.com/content/pdf/1756-0500-4-457.pdf
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