Biotechnology and Its Applications
Biotechnology and Its Applications tests the toolkit - restriction enzymes, vectors, PCR, gel electrophoresis - and then its products: Bt crops, RNAi, recombinant insulin, gene therapy, plus patents and biopiracy.
Core toolkit
- Restriction endonucleases cut palindromic sequences. EcoRI cuts G^AATTC and leaves sticky ends that can be ligated.
- A vector needs an origin of replication, a selectable marker and a cloning site. pBR322 carries amp^R and tet^R; insertion at the tet^R site inactivates it, identifying recombinants (insertional inactivation). Ti plasmid of Agrobacterium serves plants; retroviruses serve animal cells.
- PCR: denaturation near 94°C, primer annealing near 55°C, extension by Taq polymerase near 72°C. Taq survives the high temperature.
- Gel electrophoresis separates DNA by size; smaller fragments move farther.
Applications
- Bt cotton carries cry1Ac and cry2Ab from Bacillus thuringiensis. The plant makes an inactive protoxin; the alkaline insect gut solubilises it, the active toxin binds midgut cells, forms pores and kills the bollworm.
- RNAi: transgenic tobacco expressing nematode-specific double-stranded RNA silences Meloidogyne incognita mRNA and prevents infestation.
- Insulin: proinsulin's C-peptide is removed; A and B chains made separately in E. coli are joined by disulphide bonds (Humulin).
- Gene therapy for ADA deficiency: lymphocytes corrected ex vivo and returned; bone-marrow transplant is curative.
Worked example
One DNA molecule undergoes 20 PCR cycles. Copies = 2^20 ≈ 1.05 × 10^6. Reasoning: each cycle doubles the target, so after n cycles there are 2^n copies; early cycles make long products, later cycles make the short defined amplicon.
Traps that cost marks
- Saying the bacterium kills the insect; the plant expresses the gene and the insect's gut activates the protoxin.
- Using human DNA polymerase for PCR; Taq is required.
- Confusing ELISA (antibody-based detection) with PCR (DNA amplification).