A simple method for elimination of false positive results in RT-PCR

• Type: Article in International Scientific Journal
• Date: 2002

Authors

Martel, F (Author) FMUP; Grundemann, D (Author) Other; Schomig, E (Author) Other

Journal

Title: Journal of Biochemistry and Molecular Biology

Vol. 35

Pages: 248-250

ISSN: 1225-8687

Publisher: Springer

Indexing

Scopus - 21 Citations

Other information

Authenticus ID: P-007-A1K

Abstract (EN): Discrimination between the amplification of mRNA and contaminating genomic DNA is a common problem when performing a reverse transcriptase-polymerase chain reaction (RT-PCR). Even after treatment of the samples with DNAse, it is possible that negative controls (samples in which no reverse transcriptase was added) will give positive results. This indicates that there was amplification of DNA, which was not generated during the reverse transcriptase step. The possibility exists that Taq DNA polymerase acts as a reverse transcriptase, generating cDNA from RNA during the PCR step. In order to test this hypothesis, we incubated samples with a DNAse-free RNAse after the cDNA synthesis. Comparison of the results that were obtained from these samples (incubated with or without DNAse-free RNAse) confirms that the reverse transcriptase activity of Taq DNA polymerase I is a possible source of false positive results when performing RT-PCR from intronless genes. Moreover, we describe here a simple and rapid method to overcome the false positive results that originate by this activity of Taq polymerase. © BSRK & Springer-Verlag 2002.

Language: English

Type (Professor's evaluation): Scientific