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Related Experiment Videos

Reliability of PCR decontamination systems

C Niederhauser1, C Höfelein, B Wegmüller

  • 1Laboratory of Food Chemistry, Institute of Biochemistry, University of Bern, Switzerland.

PCR Methods and Applications
|October 1, 1994
PubMed
Summary

PCR contamination from degraded products causes false-negative results. While UV and uracil DNA glycosylase (UDG) prevent false positives, physical barriers are essential to maintain PCR sensitivity and avoid false negatives.

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Area of Science:

  • Molecular Biology
  • Biotechnology

Background:

  • Polymerase Chain Reaction (PCR) is susceptible to contamination from previous amplifications.
  • Accumulated intact and degraded amplicons, along with primer artifacts, can compromise subsequent PCR reactions.

Purpose of the Study:

  • To investigate the impact of degraded PCR products and primer artifacts on PCR sensitivity.
  • To evaluate methods for eliminating carryover contamination and preventing false-negative results.

Main Methods:

  • Assessed the competitive inhibition of PCR by degraded amplicons and primer artifacts.
  • Evaluated three post-PCR treatments: 8-methoxypsoralen (MOPS), hydroxylamine, and 5'-ChemiClamps.

Main Results:

  • Degraded PCR products and primer artifacts significantly decrease PCR sensitivity and can lead to false-negative outcomes.

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  • Tested post-PCR treatments (MOPS, hydroxylamine, 5'-ChemiClamps) were insufficient to inhibit artificial carryover contamination.
  • Uracil DNA glycosylase (UDG) or UV treatment effectively eliminates false-positive results.

    • Conclusions:

    • False-positive PCR results can be mitigated by UDG or UV treatment.
    • Preventing false-negative results requires physical barriers to block carryover contamination from degraded products and artifacts.