Reverse transcriptase PCR

From Wikipedia, the free encyclopedia

RT-PCR is a one or two-step process for converting RNA to DNA and the subsequent amplification of the reversely-transcribed DNA.

In the first step of RT-PCR, called the “first strand reaction,” complementary DNA (cDNA) is made from an mRNA template using dNTP’s and a reverse transcriptase. The above components are combined with a DNA primer in a reverse transcriptase buffer for an hour at 37°C.

After the reverse transcriptase reaction is complete, and cDNA has been generated from the original single-stranded mRNA, standard PCR (called the “second strand reaction”) is initiated. In the two-step RT-PCR a thermostable DNA polymerase and the upstream and downstream DNA primers are added. Heating the reaction to temperatures above 37°C facilitates binding of DNA primers to the cDNA, and 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 retro-virus and uses the enzyme HIV-Reverse Transcriptase in order to incorporate viral mRNA into genomic DNA.