Reverse transcriptase PCR

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Reverse Transcriptase Polymerase Chain Reaction, abbreviated as RT-PCR, is a two-step process for converting RNA to DNA subsequent PCR amplification of the reversely-transcribed DNA

1. first strand reaction

Complementary DNA (cDNA) is made from an mRNA template using dNTP’s & reverse transcriptase. The components are combined with a DNA primer in a reverse transcriptase buffer for an hour at 37°C.

2. second strand reaction

After the reverse transcriptase reaction is complete, cDNA has been generated from the original ss mRNA, standard PCR (called the “second strand reaction”) is initiated.

In the two-step RT-PCR a thermostable DNA polymerase & the upstream and downstream DNA primers are added. Heating the reaction to temperatures above 37°C facilitates binding of DNA primers to the cDNA, & subsequent higher temperatures allow the DNA polymerase to make double-stranded DNA from the cDNA. Heating the reaction to ~95°C melts the two DNA strands apart, enabling the primers to bind again at lower temperatures and begin the chain reaction again. After ~30 cycles, millions of copies of the sequence of interest are generated.

RT-PCR is commonly used in studying HIV, since HIV is classified as a retrovirus & uses the enzyme HIV-Reverse Transcriptase to synthesise viral DNA to be incorporated into host genome.