Recombinant DNA and PCR
To make recombinant DNA: obtain the gene by restriction enzymes (cut at specific palindromic sites, leaving sticky ends), by reverse transcriptase (mRNA → cDNA, without introns) or by synthesis. Cut a plasmid vector with the same restriction enzyme, so sticky ends are complementary; DNA ligase joins them. Add a promoter so the gene is transcribed. Transform host cells (heat shock with Ca²⁺, electroporation). Identify transformed cells with marker genes (antibiotic resistance, GFP fluorescence). PCR copies DNA: denature at 95 °C, anneal primers at ~55 °C, extend at 72 °C with Taq polymerase; each cycle doubles the DNA (). Gel electrophoresis separates DNA fragments by size: DNA (negative phosphates) moves to the anode; smaller fragments travel further.
≈ 50 minGenetic engineering applications
Recombinant human proteins made by GM bacteria, yeast or animals: insulin, factor VIII (haemophilia), adenosine deaminase; purer and more plentiful than animal sources. Gene therapy adds a working allele to treat genetic disease (e.g. SCID, cystic fibrosis) using viral vectors or liposomes; somatic therapy affects only the patient, germ-line would be inherited (banned in humans). Genetic screening detects alleles (e.g. BRCA1, CF) with DNA probes; genetic counselling helps people decide. PGD screens IVF embryos. GM crops: Bt maize (insect resistance), herbicide-tolerant soya, Golden Rice (β-carotene for vitamin A). DNA profiling uses variable repeat sequences, PCR and electrophoresis for forensics and paternity. Ethical issues: safety, gene flow to wild plants, monopolies, consent, designer babies.
≈ 45 min