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.Electronic supplementary materialThe online version of this article (doi:10.1007/s13353-015-0336-z) contains supplementary material, which is available to authorized users.
Highlights
The polymerase chain reaction, PCR, the method which has revolutionized molecular biology and diagnostics offers the advantage of exponential signal amplification (Espy et al 2006)
Respecting good laboratory practice, though it is an obvious necessity in PCR diagnostics, may be difficult to follow in molecular biology laboratories, where PCR is one of many methods in use
The method, proposed here, was used first in order to circumvent the problem of PCR contaminations occurring in the course of investigations on MutS protein (Sachadyn et al 2000; Stanisławska-Sachadyn et al 2005; StanisławskaSachadyn and Sachadyn 2005; Stanisławska-Sachadyn et al 2006; Stanisławska-Sachadyn et al 2003) where 69 bp amplicons were quantitated with real-time PCR in order to estimate the amounts of DNA bound by the protein
Summary
The polymerase chain reaction, PCR, the method which has revolutionized molecular biology and diagnostics offers the advantage of exponential signal amplification (Espy et al 2006). The unsurpassed sensitivity of the method is connected with one of the most deteriorating limitations, which is the susceptibility to the so-called contaminations leading to false positive signals (Heid et al 1996). Respecting good laboratory practices, such as, e.g., arranging separate post and pre-PCR rooms, can eliminate false positive signals resulting from PCR contaminations. The use of negative controls decreases the risk of releasing false positive. Respecting good laboratory practice, though it is an obvious necessity in PCR diagnostics, may be difficult to follow in molecular biology laboratories, where PCR is one of many methods in use. PCR contamination often acts as a silent enemy, unreported in publications, but absorbing time and generating unproductive costs in research studies
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