Biotechnology: Principles & Processes — Essential NEET Facts — Summary

Restriction enzymes are isolated from bacteria where they degrade foreign (viral) DNA — act as bacterial immune defence
EcoRI recognizes 5'-GAATTC-3' (palindromic) and cuts between G and A on both strands → sticky ends with AATT overhangs
Over 900 restriction enzymes discovered from 230+ bacterial strains
Palindromic sequence: reads the same on both strands in 5'→3' direction
Sticky ends = single-stranded overhangs; Blunt ends = flush cuts with no overhangs
DNA ligase joins DNA fragments by forming phosphodiester bonds (molecular glue)
pBR322 — prototype E. coli plasmid with: ori, ampR, tetR, and multiple cloning sites
Insertional inactivation: insert into tetR site → loss of tetracycline resistance → recombinants grow on ampicillin but NOT tetracycline
Ti plasmid from Agrobacterium tumefaciens transfers T-DNA into plant genomes; must be disarmed (tumor genes removed) before use
Competent cells = cells capable of taking up foreign DNA; made by $CaCl_{2}$ treatment + heat shock at 42 °C for 60–90 seconds
Biolistics (gene gun): DNA coated on gold or tungsten microparticles fired at high velocity — useful for plants
PCR = Polymerase Chain Reaction; invented by Kary Mullis (Nobel Prize Chemistry, 1993)
PCR cycle: Denaturation 94–98 °C → Annealing 50–65 °C → Extension 72 °C
Taq DNA polymerase from Thermus aquaticus — thermostable, not denatured during PCR heating
After n cycles of PCR: 2ⁿ copies produced (exponential amplification)
Gel electrophoresis: DNA migrates toward the anode (positive electrode) because DNA is negatively charged (phosphate backbone)
Smaller DNA fragments travel farther in agarose gel
Ethidium bromide stains DNA → orange fluorescence under UV light
Bioreactor: stirred-tank has agitator (mixing), sparger (oxygen supply)
Downstream processing: separation → purification → formulation → quality control