Reverse Transcription of RNA

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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