Reverse Transcription Polymerase Chain Reaction

Reverse transcription-polymerase chain reaction (RT-PCR) may be thought of as RNA-based PCR. RT-PCR was made possible by the discovery in the early 1970s of retroviral reverse transcriptase (RT), an RNA-dependent DNA polymerase, by David Baltimore and Howard Temin,27 for which they shared the Nobel Prize in 1975. Reverse transcriptase catalyzes DNA synthesis using RNA as the template, producing a DNA strand complementary to the RNA template, called complementary DNA (cDNA). Because cDNA is not subject to RNase degradation, it is far more stable than the corresponding RNA. Complementary DNA can be treated like any other DNA target in subsequent PCR. Logistically, RT-PCR is trivially more time-consuming than PCR due to the extra enzymatic step of reverse transcription, but there are enzymes that combine reverse transcription and DNA polymerase activities, making RT-PCR more efficient. With the introduction of techniques to successfully isolate and protect RNA from ubiquitous RNases, and to synthesize cDNA by reverse transcription and the discovery of PCR, RNA analysis is now virtually as rapid and sensitive as PCR-based DNA investigation. RT-PCR is a high-volume test method for the clinical molecular laboratory due to its use in the diagnosis and quantitation of RNA virus infections, principally HIV and HCV.

Examples of Applications of RT-PCR

1. HIV and HCV viral load determinations

2. Detection of BCR/ABL translocation diagnostic of chronic myelogenous leukemia

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