Biotechnology
Biotechnology in NIPER JEE tests recombinant DNA methods, vectors and hosts, PCR and blotting, immunotechnology, cell culture, and bioprocess/biosimilar concepts.
Core rules and concepts
- Restriction endonucleases recognize palindromic sequences; EcoRI cuts G^AATTC, producing 5′ overhangs. Ligase seals phosphodiester nicks.
- Plasmid vectors need an origin of replication, selectable marker, multiple cloning site, and small size. pBR322 carries ampR and tetR; pUC vectors use ampR and lacZ α-complementation for blue-white screening.
- PCR cycle: denaturation (~95 °C), annealing (~50–65 °C, primer Tm dependent), extension (~72 °C for Taq). Taq lacks 3′→5′ exonuclease proofreading; high-fidelity enzymes such as Pfu are used when sequence accuracy matters.
- Blotting: Southern = DNA, Northern = RNA, Western = protein. ELISA detects antigen/antibody; monoclonal antibodies come from hybridoma fusion.
- Host systems: E. coli for simple proteins; CHO and HEK293 for glycosylated biologics; yeast and Pichia for secretion.
Worked example
A PCR starts with 1,000 template copies and runs 20 complete cycles at 100% efficiency. Product copies = 1,000 × 2^20 = 1,000 × 1,048,576 ≈ 1.05 × 10^9 copies. If efficiency were only 90%, use 1,000 × (1.9)^20 ≈ 1,000 × 3.75 × 10^5 ≈ 3.8 × 10^8, showing why cycle number and efficiency must be stated.
Traps that cost marks
- Mixing up transformation (bacteria) with transfection (eukaryotic cells).
- Assuming Taq proofreads; it does not.
- Calling a selectable marker a reporter gene; lacZ is useful for screening but is not the same as antibiotic selection.
- Forgetting that Northern blotting is RNA, not protein.