Reverse transcriptase template switching during reverse transcriptase-polymerase chain reaction: Artificial generation of deletions in ribonucleotidereductase mRNA

Using reverse transcriptase-polymerase chain reaction (RT-PCR), we have rec ently described a bona tide deletion within the coding sequence of the larg e subunit of ribonucleotide reductase (R1) mRNA in colon cancer. Consecutiv e studies have raised questions about the nature of this phenomenon, becaus e the corresponding genomic alteration at the DNA level or an aberrant prot ein could not be detected. Thus we considered an in vitro artifact during R T-PCR as a possible explanation for this observation. In contrast to revers e transcriptase, Tag DNA polymerase or C. therm DNA polymerase did not gene rate the aberrant product, suggesting the demand for the template switching activity intrinsic to retroviral reverse transcriptases. In fact, virtuall y the same deletion was observed in RT-PCR experiments when in vitro transc ribed R1 mRNA was used. Considering structural prerequisites for template s witching within R1 mRNA, we show that two direct repeats adjacent to a stro ng stem-loop secondary structure flank the deleted region of 1851 base pair s. Because several mRNAs encoding proteins of clinical and diagnostic impor tance fulfill these criteria, template switching enhances the potential ris k of observing artifacts when interpreting results from RT-PCR studies. As shown in the present example, this may involve the artificial generation an d the misinterpretation of PCR fragments amplified from targets relevant to tumor biology or cancer pharmacology. As a possible solution, one-step PCR with C, therm polymerase should be considered. This polymerase eliminates the artificial generation of aberrant mRNA signals observed during cDNA syn thesis.