The human insulin gene holds two introns. To develop bacteria that produce human insulin, cDNA was the first option, as the bacteria are not capable of producing introns. The genomic DNA has introns, while cDNA is intron-free molecule, as it is the copy of processed mRNA.
The insulin gene with the two introns is transcribed in the pancreas into pre-mRNA. The introns are removed by splicing and A residues are inserted at the 3' end to produce poly adenylated mRNA. From the mRNA molecule, the cDNA can be produced with the use of reverse transcriptase, isolated from retroviruses. Just before creating cDNA, the purified mRNA is added to the enzyme reverse transcriptase. The mixture also consists of four dNTPs, and a short primer of polymerized dTTP residue called an oligo-dT primer.
The primer molecule anneals to the poly (A) tail of the mRNA being copied. By utilizing the mRNA molecule as a template, reverse transcriptase catalyzes the production of a single-stranded DNA molecule beginning from the oligo-dT primer. Then the cDNA can be copied into a double-stranded cDNA by DNA polymerase.