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cDNA Synthesis By Reverse Transcription

Normally DNA is transcribed into mRNA but this process can also be reversed. To do this we can utilize the 3’-Poly A tail which all mRNA have in common. Since this tail only contains adenosine nucleotides, we can use it as a location for our primer. We can use an Oligo (dt) Primer which contains a sequence of 12-20 thymine nucleotides, meaning that it is complementary to the 3’-Poly A tail of the mRNAs. Now Reverse Transcriptase can attach to the primed region and synthesize the complementary DNA strand in the 3’-5’ direction. After the Reverse Transcriptase is finished, we are now left with a 1/2 mRNA molecule and a 1/2 cDNA molecule. RNA nicking using RNase H solves this by creating “holes” in the RNA strand which in turn provides 3’-OH priming sites. Now DNA Polymerase 1 can fix these holes through normal DNA synthesis in the 5’-3’ direction. But in addition DNA Polymerase 1 also has exonuclease activity, meaning that it has the ability to remove and replace the remaining mRNA in the molecule! Finally, DNA Ligase can glue the molecule together, by catalyzing the formation of Phosphodiester bonds in the DNA. In this way, the mRNA is entirely replaced by cDNA. But Lucas, this seems like a bunch of extra steps, just to create DNA, I hear you screaming at your screen. I was wondering the same thing and the reason why this is still very useful is that this cDNA and DNA are not the same. Remember, this cDNA has been created only from mRNAs, which in turn have been created from DNA that was about to be expressed. In other words, this cDNA does not contain any introns or all the DNA that does not get expressed! So why is it useful to generate cDNA? Well, turns out it’s mainly 2 reasons: 1. Transcriptomics, where cDNA can be analyzed using qPCR to quantitatively determine the gene expression of an organism 2. Gene cloning, where the cDNA can be inserted into a plasmid, which in turn can be inserted into an organism that then expresses the selected gene.