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Eliminating PCR contamination: is UV irradiation the answer?
J C Fox1, M Ait-Khaled, A Webster
1Division of Communicable Diseases, Royal Free Hospital School of Medicine, London, U.K.
Journal of Virological Methods
|August 1, 1991
Summary
UV light treatment inactivates contaminating DNA in polymerase chain reaction (PCR) reagents, preventing false positives. However, this method can reduce PCR sensitivity, requiring empirical testing for each specific application.
Area of Science:
- Molecular Biology
- Virology
- Biotechnology
Background:
- High sensitivity of Polymerase Chain Reaction (PCR) leads to false positives from minimal DNA contamination.
- Contaminating DNA is a significant limitation for routine diagnostic applications of PCR.
Purpose of the Study:
- To evaluate UV light exposure as a method to eliminate contaminating DNA in PCR reagents.
- To assess the impact of UV irradiation on the sensitivity and specificity of PCR assays for viral detection.
Main Methods:
- PCR reagents were exposed to UV light before amplification.
- Assays for human cytomegalovirus (CMV) and human immunodeficiency virus (HIV) detection were tested.
- Sensitivity was determined by limiting dilution of target DNA and product analysis via gel electrophoresis.
Main Results:
- UV irradiation effectively eradicated laboratory contamination and low levels of plasmid DNA (below 100 pg).
- PCR sensitivity for HIV-1 LTR sequence amplification remained unaffected.
- PCR sensitivity for CMV early gene promoter amplification was reduced 1000-fold.
- UV treatment did not alter the size of PCR products.
Conclusions:
- UV light pre-treatment is a viable strategy to prevent false-positive PCR results caused by DNA contamination.
- The impact of UV irradiation on PCR sensitivity varies and must be empirically determined for each specific assay.
- UV treatment offers a simple method to improve the reliability of PCR diagnostics, provided sensitivity is carefully evaluated.