ONE-PAGE RAPID REVIEW — BIOTECHNOLOGY (BT-01)
RESTRICTION ENZYMES
- Molecular scissors; cut palindromic sequences in dsDNA
- EcoRI: from E. coli RY13 | Sequence: 5'-GAATTC-3' | Ends: STICKY (5'-AATT)
- Blunt ends: SmaI, EcoRV | Sticky ends: EcoRI, BamHI, HindIII
-
900 enzymes from >230 bacterial strains
- Protect bacteria by cleaving unmethylated foreign DNA (phages)
DNA LIGASE
- Molecular glue; seals phosphodiester bonds between DNA fragments
- T4 DNA ligase uses ATP; bacterial ligase uses NAD+
VECTORS
| Vector | Source | Markers | Insert Size | Special Feature |
|---|---|---|---|---|
| pBR322 | E. coli (artificial) | ampR + tetR | ~10 kb | Insertional inactivation |
| Lambda phage | Bacteriophage λ | Plaque morphology | ~23 kb | Large insert capacity |
| Ti plasmid | Agrobacterium tumefaciens | T-DNA integration | Variable | Plant transformation (dicots) |
PCR — KARY MULLIS (Nobel 1993)
| Step | Temperature | Time | Event |
|---|---|---|---|
| Denaturation | 94-98°C | 15-30 sec | dsDNA → ssDNA (H-bonds broken) |
| Annealing | 50-65°C | 20-40 sec | Primers bind template |
| Extension | 72°C | ~1 min/kb | Taq synthesizes new strand |
- Taq polymerase: from Thermus aquaticus (thermostable; survives 94°C; lacks proofreading)
- Amplification: 2^n copies after n cycles; 30 cycles ≈ 10^9 copies
GEL ELECTROPHORESIS
- DNA (negative charge) → Anode (positive electrode)
- Smaller fragments travel farther (faster through agarose pores)
- Stain: Ethidium bromide → orange bands under UV light
- Circular: n cuts = n fragments | Linear: n cuts = n+1 fragments
DNA ISOLATION
- Bacteria: Lysozyme → RNase → Protease → Ethanol precipitation
- Plants: Cellulase → RNase → Protease → Ethanol precipitation
TRANSFORMATION METHODS
| Method | Used For | Temperature |
|---|---|---|
| CaCl2 + heat shock | E. coli | 42°C, 60-90 sec |
| Electroporation | Bacteria, yeast, mammalian | Variable |
| Biolistics | Plants, organelles | N/A (physical) |
| Microinjection | Animal cells, oocytes | N/A (physical) |
BIOREACTOR
- Stirred-tank: Agitator (mixing) + Sparger (air supply) + sensors (T, pH, O2)
- Downstream: Separation → Purification → Formulation → Quality Control
CRITICAL NEET FACTS
- EcoRI → sticky (AATT) | SmaI → blunt
- DNA → ANODE (positive) | EtBr → orange bands | UV visualization
- pBR322 → ampR + tetR | Recombinant: Amp YES, Tet NO
- PCR inventor: Kary Mullis | Taq from: Thermus aquaticus
- Ti plasmid from: Agrobacterium tumefaciens | Works for: Dicots (not monocots)
- Heat shock: 42°C (not 37°C!) | PCR copies: 2^n