Biotechnology: Principles, Processes and Applications
Biotechnology questions test the toolbox (enzymes, vectors, PCR, electrophoresis) and its products (Bt crops, RNAi, insulin, gene therapy). Marks are usually lost on enzyme names, temperatures and the exact step at which a recombinant is selected.
Principles and processes
- Restriction endonucleases cut palindromic sequences; EcoRI cuts between G and A to leave 5' overhangs (sticky ends), while Hind II produces blunt ends.
- A vector needs an origin of replication, a selectable marker and a cloning site; pBR322 carries ampicillin and tetracycline resistance, and the Ti plasmid of Agrobacterium is used for plants.
- Insertional inactivation: inserting DNA into the tetracycline-resistance gene makes the recombinant ampicillin-resistant but tetracycline-sensitive.
- PCR: denaturation near 94 degrees Celsius, annealing near 50 to 55, extension at 72 with thermostable Taq polymerase from Thermus aquaticus.
- Gel electrophoresis: DNA is negatively charged and moves to the anode; smaller fragments travel farther; bands are seen with ethidium bromide under UV.
Applications
- Bt cotton carries cry1Ac and cry2Ab; the protoxin is activated in the alkaline insect gut.
- RNAi using double-stranded RNA silenced Meloidogyne incognita in tobacco.
- Golden rice is enriched in beta-carotene.
- Insulin is first made as proinsulin with C-peptide; mature insulin has A and B chains joined by disulphide bonds.
- Gene therapy for ADA deficiency uses the patient's lymphocytes with a retroviral vector.
- Molecular diagnosis uses PCR for very early detection and ELISA for antigen or antibody.
Worked example
To identify pBR322 recombinants: transform E. coli, plate on ampicillin so all transformants grow, then replica-plate on tetracycline. Colonies that fail to grow on tetracycline carry an insert inside the tetracycline-resistance gene and are the recombinants.
Traps that cost marks
- Taq polymerase is thermostable; ordinary DNA polymerase denatures in PCR.
- The Bt gene is cry, not a generic Bt gene.
- Insulin's C-peptide is removed during maturation.
- ADA gene therapy uses lymphocytes, not a direct injection of naked DNA.