title: A simple modification of PCR thermal profile applied to evade persisting contamination.
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.
authors.affiliation: Department of Molecular Biotechnology and Microbiology, Gdańsk University of Technology, Gdańsk, Poland.
authors.forename: Michał
authors.initials: M
authors.lastname: Banasik
authors.affiliation: Department of Biology and Genetics, Medical University of Gdańsk, Gdańsk, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Stanisławska-Sachadyn
authors.affiliation: Department of Molecular Biotechnology and Microbiology, Gdańsk University of Technology, Gdańsk, Poland. psach@pg.gda.pl.
authors.forename: Paweł
authors.initials: P
authors.lastname: Sachadyn
mesh_terms: D059018:DNA Contamination; D005189:False Positive Reactions; D016133:Polymerase Chain Reaction / Q000379:methods*; D013696:Temperature
keywords: False positive signal; No-template control; PCR contamination; Quantitative PCR; qPCR
references.citation: Clin Microbiol Rev. 2006 Jan;19(1):165-256
references.pmid: 16418529
references.citation: Genome Res. 1996 Oct;6(10):986-94
references.pmid: 8908518
references.citation: Mutat Res. 2005 Feb 15;570(1):97-103
references.pmid: 15680407
references.citation: J Biotechnol. 2006 Jan 24;121(2):134-43
references.pmid: 16157410
references.citation: Nucleic Acids Res. 2000 Apr 15;28(8):E36
references.pmid: 10734213
references.citation: PCR Methods Appl. 1993 Oct;3(2):S2-7
references.pmid: 8268784
references.citation: Protein Expr Purif. 2003 Mar;28(1):69-77
references.pmid: 12651109
references.citation: Acta Biochim Pol. 2005;52(3):575-83
references.pmid: 16082411