(i) Isolation of Insulin Genes: The first step involves isolating the genes that code for the two polypeptide chains of insulin, known as Chain A and Chain B. These genes are typically obtained from human DNA.

(ii) Use of Restriction Enzymes:Once the genes are isolated, restriction enzymes are employed to cut both the insulin genes and the vector DNA (usually a plasmid) at specific sequences. This creates compatible ends for the next step.

(iii) Insertion into Vector DNA:The next step is to insert the isolated insulin genes into the plasmid vector. This is done by mixing the cut insulin genes with the cut plasmid DNA. The two DNA fragments are then joined together using an enzyme called DNA ligase.

(iv) Transformation into Host Cells:The recombinant DNA (plasmid containing the insulin genes) is then introduced into a suitable host organism, commonly E. coli. This process is known as transformation, where the bacteria take up the plasmid.

(v) Replication of Recombinant DNA: Inside the E. coli, the recombinant DNA replicates as the bacteria divide. This leads to the production of multiple copies of the insulin genes.

(vi) Extraction of Insulin Chains:After sufficient growth, the E. coli cells are harvested, and the insulin chains (A and B) are extracted from the bacterial cells.

(vii) Joining the Chains: Finally, the extracted chains A and B are chemically linked together by forming disulfide bonds, resulting in the formation of functional human insulin.