Reverse transcription of RNA is a process whereby RNA, typically messenger RNA is converted into complimentary DNA. The process was discovered by Howard Temin and John Baltimore when they observed that certain RNA viruses could revert to DNA. This was an important discovery that led to the discovery of enzymes classified as reverse transcriptases. Today Reverse Transcription is routinely applied in molecular biology laboratories to obtain the stable cDNA version of RNA for downstream analysis.

1. REVERSE TRANSCRIPTION OF RNA
KENNETH F. RODRIGUES, PHD

2. HOWARD MARTIN TEMIN & DAVID BALTIMORE
Discovered the process of reverse transcription and the enzyme reverse transcriptase
By Bob Paz (Bob Paz) [CC BY-SA 3.0 (https://creativecommons.org/licenses/by-
sa/3.0) or GFDL (http://www.gnu.org/copyleft/fdl.html)], via Wikimedia Commons

3. REVERSE TRANSCRIPTASE
Image source: https://commons.wikimedia.org/wiki/File:Reverse_Transcriptase_1HMV.png
The enzyme reverse transcriptase is a RNA dependent DNA polymerase

4. THE PROCESS OF REVERSE TRANSCRIPTION
 Annealing of the primer to the RNA transcript OR to the poly A tail.
 Reverse transcription by Reverse Transcriptase.
 Removal of mismatches by RNase H.
 Repair of gaps by DNA Polymerase I, Klenow fragment.
 Downstream processing of cDNA

5. ENZYMES EMPLOYED FOR REVERSE TRANSCRIPTION
 Reverse Transcriptase
 RNase H
 DNA Polymerase I (Klenow fragment)

6. STEP 1: ANNEALING OF THE PRIMER
 RNA tends to form secondary structures via Watson-Crick base
pairing, the first step involves denaturation of the RNA by heating
in the presence of the gene specific primer followed by cooling to
room temperature to facilitate primer annealing.
5’ 3’

7. STEP 2: REVERSE TRANSCRIPTION
 Reverse transcriptase commences transcription at the primer
binding site.
5’ 3’

8. STEP 3: REMOVAL OF MISMATCHES BY RNASE H
 The mismatches in the DNA:RNA hybrid generated by the Reverse
Transcriptase are removed by the enzyme RNase H.
5’ 3’

9. STEP 4: REPAIR OF STRAND BY DNA POLYMERASE I KF
 DNA Polymerase I (Klenow Fragment ) repairs the gaps and fills in
nucleotides leading to the formation of a double stranded DNA.

10. DOWNSTREAM PROCESSING
 The resulting DNA strand can be subjected to DNA sequencing in
order to determine the identity of the transcript.
 The copy number of the transcripts can be determined using real
time PCR.

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