Investigating CSI – Background material Table of Contents I ...

Real-Time Polymerase Chain Reaction (PCR)
Polymerase chain reaction (PCR) allows the logarithmic copying of part of
a DNA molecule using a DNA polymerase enzyme that is tolerant to
elevated temperatures. RT PCR uses mRNA as a template to make
DNA. The steps involved are discussed below:
1. mRNA is copied to cDNA (chromosomal DNA) by reverse transcriptase
using an oligo dT primer (random oligomers may also be used). In realtime
PCR, we usually use a reverse transcriptase that has an endo H
activity. This removes the mRNA allowing the second strand of DNA to be
formed. A PCR mix is then set up which includes a heat-stable
polymerase (such as Taq polymerase), specific primers for the gene of
interest, deoxynucleotides and a suitable buffer.
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