Reverse PCR – cDNA Synthesis

DNA is suitable for diagnostic purposes when the infectious agent is a bacterium, a parasite or a DNA virus. A large proportion of viruses are RNA viruses, however, i.e. their genomes consist of RNA and are replicated in host cells as RNA. Reverse transcription of RNA to complementary DNA (cDNA) is therefore an obligatory step for the identification of RNA viruses. When PCR DNA amplification is preceded by cDNA synthesis, the combined protocol is named reverse PCR. Reverse PCR may also be useful in order to quantify transcription (mRNA synthesis) to show the activity of an infectious genome. Using enzymes such as rTth, which has both reverse transcriptase and DNA polymerase activity and is thermostable, reverse transcription can be achieved in one reaction and one tube.
Otherwise, the reverse transcription is often carried out as a separate first step using a thermolabile reverse transcriptase (typically cloned retroviral enzymes). If random primers (hexamers or nonamers) or oligo-dT primers are used in the initial reverse transcription, then the subsequent PCR can be performed with different specific primer pairs and designed for different RNA viruses. According to the rTth reverse PCR, the same reverse primer sequence will prime both cDNA synthesis and the PCR amplification. This may increase specificity, but reduces the versatility compared with random hexamer or oligo-dT primed cDNA.

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