Biotechnology uses living organisms or their components to develop products, and recombinant DNA technology is its central tool for NEET — combining DNA from different sources and expressing it in host cells.

Restriction endonucleases are bacterial enzymes that cut DNA at specific palindromic sequences; EcoRI recognizes 5'-GAATTC-3' and makes a staggered cut that produces sticky ends with 4-nucleotide overhangs.

DNA ligase joins DNA fragments by sealing phosphodiester bonds between the sugar-phosphate backbones, acting as the molecular glue that creates a continuous recombinant DNA molecule.

The plasmid pBR322 is the prototype cloning vector with four key features: origin of replication, ampicillin resistance, tetracycline resistance, and multiple restriction sites for foreign DNA insertion.

Insertional inactivation identifies recombinant colonies: when DNA is inserted into the tetR gene, tetracycline resistance is lost, so recombinants grow on ampicillin plates but not on tetracycline plates.

The Ti plasmid from Agrobacterium tumefaciens transfers T-DNA into plant genomes and must be disarmed (tumor genes removed) before use as a vector in plant genetic engineering.

Bacterial transformation requires competent cells, achieved by $CaCl_{2}$ treatment followed by heat shock at 42 °C for 60–90 seconds; biolistics $\frac{gene gun with gold}{tungsten particles}$ is used for plant cells.

PCR (invented by Kary Mullis) amplifies DNA in vitro through three thermally cycled steps: denaturation at 94–98 °C, annealing at 50–65 °C, and extension at 72 °C using thermostable Taq polymerase from Thermus aquaticus.

After n cycles of PCR, 2ⁿ copies are produced; gel electrophoresis then separates these fragments through agarose gel — DNA migrates toward the anode because it is negatively charged, and smaller fragments travel farther.

Stirred-tank bioreactors with agitators and spargers enable large-scale production of recombinant proteins, followed by downstream processing (separation, purification, formulation, quality control) to yield the final therapeutic product.