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False negative results from using common PCR reagents

Bacich, DJ and Sobek, KM and Cummings, JL and Atwood, AA and O'Keefe, DS (2011) False negative results from using common PCR reagents. BMC Research Notes, 4.

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Background: 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 60 ng 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. © 2011 O'Keefe et al; licensee BioMed Central Ltd.


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Item Type: Article
Status: Published
CreatorsEmailPitt UsernameORCID
Bacich, DJ
Sobek, KM
Cummings, JLjld247@pitt.eduJLD247
Atwood, AA
O'Keefe, DS
Date: 28 October 2011
Date Type: Publication
Journal or Publication Title: BMC Research Notes
Volume: 4
DOI or Unique Handle: 10.1186/1756-0500-4-457
Schools and Programs: School of Medicine > Urology
Refereed: Yes
Date Deposited: 26 Oct 2016 17:11
Last Modified: 29 Jan 2019 15:56


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