Biomics

The introduction of thermostable Taq DNA polymerase from the thermophilic eubacterium Thermus aquaticus into PCR has transformed the method into a widely used tool for a variety of applications, including the assessment of transcriptional activity of individual genes and the detection of RNA viruses. These applications required a preliminary step of converting RNA molecules into complementary DNA (cDNA), which was initially performed exclusively using viral reverse transcriptases. However, the thermolability of these enzymes created significant limitations, as both reverse transcription and subsequent PCR had to be carried out under suboptimal conditions. In addition, at lower temperatures, RNA molecules tend to form stable secondary structures that hinder efficient cDNA synthesis. In this context, it was highly attractive to use thermostable Taq DNA polymerase directly for cDNA synthesis, and this approach proved successful. It was subsequently found that Tth DNA polymerase from the related bacterium T. thermophilus, in the presence of manganese ions, exhibits reverse transcriptase activity that is approximately two orders of magnitude higher than that of Taq polymerase. The subsequent development of genetically engineered thermostable DNA polymerases based on Taq, Tth polymerases, and several other similar enzymes has significantly improved the reverse transcription step. Importantly, some of these variants successfully combine reverse transcriptase activity with proofreading 3′→5′ exonuclease activity, thereby reducing the error rate during cDNA synthesis.

thermostable DNA polymerase, Taq polymerase, Tth polymerase, reverse transcriptase activity, RT-PCR, cDNA

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