Using reverse transcriptase to isolate a gene

• Firstly, a cell that readily produces the protein is selected (e.g. the beta cells of the Islets of Langerhans from the pancreas are used to produce insulin).
• The cells selected have large quantities of the relevant mRNA, which is therefore more easily extracted. Reverse transcriptase is then used to make DNA from RNA.
• The DNA produced from RNA (using the reverse transcriptase enzyme) is known as complementary DNA (cDNA) because it is made up of nucleotides that are complementary to the mRNA.
• To make up the second strand of the synthesised DNA, the enzyme DNA polymerase is used to build up the complementary nucleotides (primers) on the cDNA template. These will anneal to their complementary bases using hydrogen bonds, and the nucleotides will be joined by phosphodiester bonds. This forms double-stranded DNA.
• DNA Polymerase catalyses condensation reactions to join together adjacent nucleotides.