Methods Used in Recombinant DNA Technology
There are several methods used to create recombinant DNA molecules and genetically modified organisms. The key methods involved are:
- Restriction Enzyme Digestion: Restriction enzymes are used to cut DNA molecules at specific DNA sequences known as restriction sites. This generates fragments of DNA with sticky or blunt ends that allow joining of DNA from multiple sources.
- Ligation: DNA ligase is used to join DNA fragments together by catalyzing the formation of phosphodiester bonds between the 3'-hydroxyl and 5'-phosphate ends of DNA fragments. This permanently joins the DNA strands together.
- Polymerase Chain Reaction (PCR): PCR is used to amplify specific target DNA fragments in vitro. It involves repeated thermal cycling. This allows mass production of specific DNA sequences from even small starting amounts of DNA sample.
- cDNA Synthesis: mRNA extracted from cells is reverse transcribed into its complementary DNA strand (cDNA) using the enzyme reverse transcriptase. This cDNA can then be used like regular DNA in genetic engineering applications.

Applications of Recombinant DNA Technology
Some key applications of Recombinant DNA Technology  include:
- Production of Human Insulin: Recombinant human insulin was one of the earliest products of genetic engineering. It is now used to treat millions of diabetics globally by substituting animal-derived insulin.
- Vaccine Development: Recombinant subunit vaccines against Hepatitis B, Pertussis, Human Papillomavirus etc have been developed using this technology by isolating protective antigen genes.
- Development of Herbicide and Pest Resistant Crops: Genes from bacteria like Bacillus thuringiensis have been engineered into crops like corn, cotton, soya and brinjal to make them resistant against fungal, viral and insect pests.
- Production of Therapeutic Proteins: Many biopharmaceuticals like somatropin (growth hormone), erythropoietin, tissue plasminogen activator etc are now commercially produced through the expression of recombinant versions in bacterial or mammalian cell cultures.
- Gene Therapy: This technology holds promise to treat genetic diseases by introducing functional copies of missing or defective genes into patient's cells and tissues. Though efficacy and safety challenges need to be overcome.

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