Reverse Transcriptase PCRReverse Transcriptase PCR or RT – PCR is a one or two step process that is undergone when converting RNA to DNA and the subsequent amplification of the DNA that has been reversely transcribed.
In the first step of RT PCR, which is called the first strand reaction, the complementary DNA (cDNA) is produced from an mRNA template that uses dNTPs and a reverse transcriptase. The components mentioned are combined with a DNA primer in a reverse transcriptase buffer for approximately one hour at a temperature of 37 degrees Celsius.
When the reverse transcriptase reaction has been completed a cDNA will have been produced from the original single stranded mRNA. At this point standard PCR, or second strand reaction, is performed. In this two step RT – PCR a thermostable DNA polymerase and the upstream and downstream DNA primers are added. The reaction is further helped by putting it in a temperature that is above 37 degrees C will aid in binding DNA primers to the cDNA. The subsequent higher temperatures will let the DNA polymerase to produce double stranded DNA from the cDNA.
RT – PCR is most commonly utilized in studying HIV because HIV is classified as a retro virus and thus uses the enzyme HIV – Reverse Transcriptase in order to mix in viral mRNA into genomic DNA.
Web Resources On Reverse Transcriptase PCR
RT-PCR: The Basics
Book Resources On Reverse Transcriptase PCRBiology of Negative Strand RNA Viruses: The Power of Reverse Genetics by Yoshihiro Kawaoka
A History of Genetics by A. H. Sturtevant, Edward B. Lewis
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