Biotechnology and Its Applications: Tools, Processes and Products
Biotechnology and Its Applications tests the tools of recombinant DNA technology, the processes used to make products, and specific applications in medicine, agriculture and diagnostics.
Tools and technique
- Restriction endonucleases cut palindromic DNA sequences and leave sticky ends; EcoRI cuts G↓AATTC. DNA ligase joins fragments.
- A vector needs an origin of replication, a selectable marker (ampR, tetR) and unique restriction sites; pBR322 is the standard example.
- Insertional inactivation: an insert inside tetR makes recombinants tetracycline-sensitive but ampicillin-resistant.
- Gel electrophoresis separates DNA by size; DNA is negatively charged and moves toward the anode.
PCR and scale-up
- PCR: denaturation near 94 °C, primer annealing near 50–60 °C, extension at 72 °C by thermostable Taq polymerase.
- Stirred-tank bioreactors maintain oxygen, pH and temperature for large-scale culture; downstream processing purifies the product.
Worked example
Starting from one DNA molecule, how many copies are present after 30 PCR cycles? Each cycle doubles the number, so copies = 2³⁰ ≈ 1.07 × 10⁹ - about a billion copies, which is why PCR detects trace DNA.
Applications
- Human insulin: A and B chains are produced separately in E. coli and then joined, with the C-peptide removed; Eli Lilly marketed it as Humulin in 1983.
- Bt cotton carries cryIAc and cryIIAb genes from Bacillus thuringiensis against bollworm; the protoxin is activated by the alkaline insect gut.
- RNAi against Meloidogyne incognita; gene therapy for ADA deficiency; ELISA and PCR for early diagnosis.
Traps
- Taq polymerase is used because it survives high temperature.
- Bt toxin is a protein, so the gene comes from bacteria, not from a spray.
- Gene therapy corrects somatic cells and is not inherited.