Reverse transcriptase template switching during reverse transcriptase–polymerase chain reaction: Artificial generation of deletions in ribonucleotide reductase mRNA

摘要: Using reverse transcriptase-polymerase chain reaction (RT-PCR), we have recently described a bona fide deletion within the coding sequence of large subunit ribonucleotide reductase (R1) mRNA in colon cancer. Consecutive studies raised questions about nature this phenomenon, because corresponding genomic alteration at DNA level or an aberrant protein could not be detected. Thus considered vitro artifact during RT-PCR as possible explanation for observation. In contrast to transcriptase, Taq polymerase C. therm did generate product, suggesting demand template switching activity intrinsic retroviral transcriptases. fact, virtually same was observed experiments when transcribed R1 used. Considering structural prerequisites mRNA, show that two direct repeats adjacent strong stem-loop secondary structure flank deleted region 1851 base pairs. Because several mRNAs encoding proteins clinical and diagnostic importance fulfill these criteria, enhances potential risk observing artifacts interpreting results from studies. As shown present example, may involve artificial generation misinterpretation PCR fragments amplified targets relevant tumor biology cancer pharmacology. solution, one-step with should considered. This eliminates signals cDNA synthesis.