Reverse transcriptase PCR

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 dNTPs & 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.