Reverse Transcriptase PCR

 REVERSE TRANSCRIPTASE PCR

Reverse transcriptase PCR (RT-PCR) uses the enzyme reverse transcriptase to make a cDNA copy of mRNA from an organism, and then uses PCR to amplify the cDNA (Fig. 4.26). The advantage of this technique is evident when trying to use PCR to amplify a gene from eukaryotic DNA. Eukaryotes have introns, some extremely long, which interrupt the coding segments. After transcription, the primary RNA transcript is processed to remove all the introns, hence becoming mRNA. Using mRNA as the source of the target DNA relies on the cell removing the introns. In practice, RT-PCR has two steps. First, reverse transcriptase recognizes the 3′ end of primers containing repeated thymines and synthesizes a DNA strand that is complementary to the mRNA.


(The thymines base-pair with the poly(A) tail of mRNA.) Then the RNA strand is replaced with another DNA strand, leaving a double-stranded DNA (i.e., the cDNA).

 

Next, the cDNA is amplified using a normal PCR reaction containing appropriate primers (one usually recognizes the poly(A) tail), Taq polymerase, and nucleotides.

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