Restriction enzymes (RE): "Molecular scissors" — cut DNA at specific palindromic sequences. Named by genus+species+strain: EcoRI (E. coli strain R), HindIII (H. influenzae strain d). Type II REs cut within or near recognition site. Produce sticky ends (complementary overhangs) or blunt ends.
Cloning vectors: Carry insert DNA into host. Plasmid pBR322 has: ori (origin of replication), ampicillin resistance gene (amp^R), tetracycline resistance gene (tet^R), restriction sites within tet^R for insertion. Bacteriophage λ can carry larger inserts. BAC (Bacterial Artificial Chromosomes) — very large inserts for genome projects.
Competent cells: Bacteria made permeable to take up DNA (heat shock + divalent cations like CaCl₂, or electroporation).
PCR (Polymerase Chain Reaction): Amplifies specific DNA sequences exponentially. Three steps: (1) Denaturation (94°C — strands separate), (2) Annealing (50–65°C — primers bind), (3) Extension (72°C — Taq polymerase extends). Uses Taq polymerase (heat-stable, from Thermus aquaticus). 30 cycles → 2³⁰ copies.
Gel electrophoresis: Separates DNA fragments by size. Agarose gel + electric field. Smaller fragments move faster (further from well). Ethidium bromide staining → orange bands under UV. Southern blotting: DNA; Northern blotting: RNA; Western blotting: Protein.
Insulin: Eli Lilly (1982) — first rDNA product. Human insulin gene inserted into E. coli plasmid. A and B chains produced separately → combined with disulphide bonds. Humulin = recombinant human insulin.
Bt toxin crops: Bacillus thuringiensis produces Cry proteins (crystalline proteins) that are toxic to specific insects. cry1Ac/cry2Ab → cotton bollworm. cry1Ab → corn borer. Bt cotton, Bt brinjal. Toxin activated only in alkaline insect gut (not in human stomach).
Golden rice: Contains beta-carotene biosynthesis genes (from daffodil + Erwinia). Provides Vitamin A precursor. Addresses Vitamin A deficiency in developing countries.
Gene therapy: Replacing/correcting defective genes. First successful: ADA deficiency (adenosine deaminase). Lymphocytes extracted, ADA gene introduced → cells returned to patient.
Biosafety levels: BSL 1 (minimal risk) to BSL 4 (highest risk, special containment required).
GMO concerns: Allergenicity, gene flow to wild relatives, antibiotic resistance marker genes, biodiversity loss.
GEAC: Genetic Engineering Approval Committee (India) — regulatory body for GMO safety assessment.
Biopiracy: Unauthorised use of biological resources/traditional knowledge. India's disputes: Neem (Azadirachta indica), Haldi (turmeric), Basmati rice patents. Biological Diversity Act 2002 — India's protection.
BamHI: cuts G↓GATCC (sticky ends)
HaeIII: GG↓CC (blunt ends)
SmaI: CCC↓GGG (blunt ends)
Palindromic sequences: read same 5'→3' on both strands
Primers (forward + reverse)
Taq polymerase (72°C optimum)
dNTPs (dATP, dGTP, dCTP, dTTP)
Mg²⁺ (cofactor for Taq)
Buffer system
Northern blotting: RNA
Western blotting: Protein
Eastern blotting: Lipids/carbs
Probe: labelled complementary sequence used for detection
Golden rice: beta-carotene genes
Rosie (cow): human alpha-lactalbumin in milk
Flavr Savr tomato: delayed ripening
ADA gene therapy: first successful gene therapy
Restriction endonucleases (Type II) cut double-stranded DNA at specific palindromic recognition sites. They are called molecular scissors. DNA ligase joins DNA fragments (molecular glue). Together they are the core tools of rDNA technology.
Bt toxin is in an inactive pro-toxin (crystal protein) form. In the alkaline gut of insects, it is activated and binds to specific receptors → lysis of cells lining the gut. Human stomach is acidic → toxin not activated. Additionally, humans lack the specific receptor. Both reasons contribute to safety.
PCR/blotting: 1 Q/year
Applications (Bt, insulin): 1 Q/year
Expected: Cry protein specificity
Watch: Gene therapy steps (ADA)