Question

In RT-PCR, what does the enzyme reverse transcriptase do? Why is this step necessary for subsequent detection of the virus using PCR?

Answer 1

RT-PCR stands for Reverse Transcription Polymerase Chain Reaction, which allows the use of RNA as the template strand. According to central dogma of molecular biology, DNA is copied into RNA by a process called transcription. In this technique, the RNA is reverse transcribed to complementary DNA (cDNA) using the enzyme reverse transcriptase. The sample taken in RT-PCR is RNA and in the first step of RT-PCR a DNA strand is formed on this RNA by the principle of complementarity. The RNA portion of this RNA-DNA hybrid is then degraded by the RNase H function of the reverse transcriptase enzyme. The remaining single stranded DNA is then converted to double stranded cDNA by the DNA-dependent DNA polymerase activity of the reverse transcriptase enzyme. From here on, the usual PCR process proceeds to amplify this sequence of cDNA- These are the roles of reverse transcriptase enzyme in RT-PCR.

Some viruses have DNA as their genetic material whereas some others have RNA as their genetic material. Polymerase chain reaction (PCR) is a technique used to amplify DNA into billions of copies. Viruses, with RNA as genetic material (such as corona virus, zika virus, influenza virus etc), cannot be detected or amplified using PCR without modifying it. PCR involves the use of a DNA-dependent DNA polymerase enzyme. So this virus RNA or genome needs to be converted to DNA (so PCR become modified into RT-PCR). As we discussed earlier this step is carried out by the reverse transcriptase enzyme. So without this step, it is not possible to detect viruses with RNA using the technique PCR.