Biotechnology: Principles and ProcessesNEET Botany · Class 12 · NCERT Chapter 7

Biotechnology: Principles and Processes contributes 3 to 5 questions per NEET paper. High-yield topics include: restriction enzymes (types, palindromic sequences, sticky vs blunt ends, naming convention), PCR (steps — denaturation, annealing, extension; Taq polymerase), gel electrophoresis (principle, EtBr staining, UV visualization), cloning vectors (pBR322 features, insertional inactivation), and competent cell transformation methods.

Restriction endonucleases are enzymes that cut double-stranded DNA at specific recognition sequences (restriction sites). They were called "molecular scissors" by Werner Arber. A palindromic sequence in molecular biology is a sequence where the base sequence on one strand reads the same as the complementary strand read in the opposite direction (5' to 3'). Example: EcoRI recognises 5'-GAATTC-3' and cuts between G and A on each strand, producing sticky (cohesive) ends with 4-nucleotide overhangs (5'-AATT-3'). Sticky ends allow compatible fragments to join. SmaI (5'-CCCGGG-3') cuts in the centre producing blunt ends.

PCR (Polymerase Chain Reaction) amplifies a specific DNA segment in vitro using three repeating steps: (1) Denaturation: heat at 94-98°C to separate double-stranded DNA into two single strands. (2) Annealing: cool to 50-65°C so specific primers bind (anneal) to their complementary template sequences. Two primers define the region to be amplified. (3) Extension (Elongation): heat to 72°C where Taq polymerase (a thermostable DNA polymerase from Thermus aquaticus bacterium) synthesises new DNA from the primer in 5' to 3' direction. Each complete cycle doubles the target DNA. After n cycles, 2^n copies are produced. After 30 cycles: 2^30 ≈ 10^9 fold amplification.

pBR322 is a plasmid cloning vector that has two antibiotic resistance genes: ampR (ampicillin resistance) and tetR (tetracycline resistance), plus multiple restriction sites within tetR. When a foreign DNA fragment is inserted into a restriction site within tetR, it disrupts the tetR gene (making the bacterium tetracycline-sensitive). But the ampR gene remains intact. So: cells with recombinant plasmid = ampR (survive ampicillin) but tetS (killed by tetracycline). Cells with non-recombinant plasmid = ampR AND tetR. By first plating on ampicillin (selects for transformed cells) then replica plating on tetracycline (recombinants cannot grow), you can identify which colonies carry the insert. This is insertional inactivation used for selection of recombinants.

In bioprocess engineering (production of biologicals using microbes or cells in bioreactors): Upstream processing includes all steps BEFORE the fermentation/production step: preparation of nutrient medium, sterilisation (autoclaving, filtration), preparation of seed culture, inoculation of the bioreactor. Downstream processing includes all steps AFTER fermentation to obtain the final purified product: separation of biomass (centrifugation, filtration), product extraction, purification (chromatography, precipitation, ultrafiltration), formulation, quality control, and packaging. Downstream processing is often the most expensive part of bioprocess manufacturing.

Gel electrophoresis separates DNA fragments by size. DNA is negatively charged (due to phosphate groups), so it migrates toward the positive electrode (anode) when an electric field is applied. Agarose gel acts as a molecular sieve: smaller DNA fragments pass through the pores more easily and travel faster (further from the well). Larger fragments move slower (stay closer to well). After electrophoresis, the gel is stained with ethidium bromide (EtBr), a fluorescent dye that intercalates between DNA bases. When viewed under UV light, EtBr-DNA complexes fluoresce orange-red, revealing bands at specific positions. A DNA ladder (size marker with fragments of known sizes) is run alongside to determine the sizes of unknown fragments.