A simple method for elimination of false positive results in RT-PCR.
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.
Martel
Fátima
F
Department of Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany. fmartel@med.up.pt
Gründemann
Dirk
D
Schömig
Edgar
E
D000818:Animals; D004247:DNA / Q000235:genetics; D003851:Deoxyribonucleases; D005189:False Positive Reactions; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D012194:RNA-Directed DNA Polymerase; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012260:Ribonucleases; D019914:Taq Polymerase
D012333:RNA, Messenger; D004247:DNA; D019914:Taq Polymerase; D012194:RNA-Directed DNA Polymerase; D003851:Deoxyribonucleases; D012260:Ribonucleases