Reverse Transcription

Mechanism of reverse transcription 10 Stages

The entire process is catalyzed by reverse transcriptase which has both DNA polymerase and RNase H activities.

    Stages 1 - 6

  1. A retrovirus-specific cellular tRNA hybridizes with a complementary region called the primer-binding site (PBS).
  2. A DNA segment is extended from tRNA based on the sequence of the retroviral genomic RNA.
  3. The viral R and U5 sequences are removed by RNase H.
  4. First jump: DNA hybridizes with the remaining R sequence at the 3' end.
  5. A DNA strand is extended from the 3' end.
  6. Most viral RNA is removed by RNase H.

  7. Stages 7 - 10

  8. A second DNA strand is extended from the viral RNA.
  9. Both tRNA and the remaining viral RNA are removed by RNase H.
  10. Second jump: The PBS region of the second strand hybridizes with the PBS region of the first strand.
  11. Extension on both DNA strands. LTR stands for "long terminal repeat".

Reproduction of HIV, a retrovirus An Overview

Reverse Transcription PCR

Reverse transcriptase polymerase chain reaction (RT-PCR) is a process whereby DNA is amplified from RNA.1 It is an extremely useful technique in RNA quantifying, cloning, cDNA library construction and signal amplification.
The technique consists of two main steps:
(1)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. There are two common reverse transcriptases. These components are combined with a DNA primer in a reverse transcriptase buffer for an hour at 37C. There are three types of DNA primers commonly used to facilitate reverse transcription.
(2)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 original reaction tube after addition of a thermostable DNA polymerase, e.g., Taq polymerase, and upstream and downstream DNA primers. Heating the reaction to temperatures above 37C 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 ~95C 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.