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Published on: May 22, 2012
A simple modification of PCR thermal profile applied to evade persisting contamination
Michał Banasik1, Anna Stanisławska-Sachadyn2, Paweł Sachadyn3
1Department of Molecular Biotechnology and Microbiology, Gdańsk University of Technology, Gdańsk, Poland.
Abstract:
The polymerase chain reaction (PCR), one of the most commonly applied methods of diagnostics and molecular biology has a frustrating downside known as the false positive signal or contamination. Several solutions to avoid and to eliminate PCR contaminations have been worked out to date but the implementation of these solutions to laboratory practice may be laborious and time consuming. A simple approach to circumvent the problem of persisting PCR contamination is reported. The principle of this approach lies in shortening the steps of denaturation, annealing, and elongation in the PCR thermal cycle. The modification leads to the radical decline of false positive signals obtained for the no-template controls without affecting the detection of target PCR products. In the model experiments presented here, the signal of negative control was shifted by about ten cycles up above those for the examined samples so that it could be neglected. We do not recommend this solution in PCR diagnostics, where the sensitivity of detection is of the highest priority. However, the approach could be useful to pass by the problem of persisting contamination in quantitative PCR, where the range of quantitation is usually much above the limits of detection.
Insights
Shortening polymerase chain reaction (PCR) thermal cycles significantly reduces false positive signals in no-template controls. This simple method avoids laborious contamination solutions without impacting target detection, proving useful for quantitative PCR applications.
Area of Science:
- Molecular Biology
- Biotechnology
Background:
- Polymerase chain reaction (PCR) is a vital molecular biology technique.
- False positive signals and contamination are significant challenges in PCR applications.
- Existing solutions for PCR contamination can be labor-intensive and time-consuming.
Purpose of the Study:
- To introduce a simple method to circumvent persistent PCR contamination.
- To evaluate the effectiveness of shortened PCR thermal cycles in reducing false positives.
- To assess the impact of this modification on target PCR product detection.
Main Methods:
- Implementing a modified PCR protocol with shortened denaturation, annealing, and elongation steps.
- Utilizing no-template controls (NTCs) to assess contamination levels.
- Comparing results with standard PCR protocols.
Main Results:
- A radical decline in false positive signals was observed in no-template controls.
- The modification did not affect the detection of target PCR products.
- Negative control signals were shifted by approximately ten cycles, rendering them negligible.
Conclusions:
- Shortened PCR thermal cycles offer a simple and effective approach to mitigate contamination issues.
- This method is particularly beneficial for quantitative PCR where high quantitation ranges are common.
- The approach is not recommended for diagnostic PCR where maximal sensitivity is critical.
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