What is complementary DNA (cDNA)
cDNA (complementary DNA) refers to the single-stranded DNA that is generated through the reverse transcription of messenger RNA templates. The enzyme known as reverse transcriptase is what makes the process of reverse transcription possible. cDNA can also be generated in retroviruses during the process of the RNA genome being converted into DNA.
The primary application for cDNA is the cloning of eukaryotic genes in prokaryotic organisms. Introns are sections of eukaryotic genes that are located in between exons and are responsible for the coding of proteins.
Figure 1: Generation of a complementary DNA (cDNA) from an isolated eukaryotic gene.
During the process of transcription, introns as well as exons are responsible for the coding of messenger RNA (mRNA). But the introns are taken out of mRNA and the exons are joined together to make a mature mRNA. The transcriptome is the sum of all the mRNA in an organism. This mRNA can be used to make cDNA, which is made up of only the parts of the genome that code for proteins.
Production of complementary DNA
Because RNA is so easily damaged by the omnipresent RNases, working with the coding sequence is much easier with complementary DNA strand than mRNA. As a result, instead of analysing the mRNA, cDNA sequences are used. For DNA microarrays, mRNA is converted into cDNA, which is used to make the probes. There are a number of steps involved in the production of cDNA. Let’s see what is required to produce cDNA.
Figure 2. Four basic reagents needed to produce cDNA: mRNA as template, dNTPs, reverse transcriptase and primers.
Once the mRNA is isolated, you need dNTPs (dGTP, dCTP, dATP, and dTTP), primers, and a DNA polymerase called reverse transcriptase.
Combine the mRNA with the other reagents, and then wait for the polymerase to finish producing a strand of DNA that is complementary to the original (first strand synthesis). Next, the mRNA must be removed and the second strand of DNA synthesized.