Learning Objectives
- Understand the principles of genetic engineering and recombinant DNA technology
- Learn about restriction enzymes, vectors, and cloning
- Study the steps in rDNA technology
- Understand PCR, gel electrophoresis, and other molecular tools
Key Concepts
Principles of Biotechnology
Two core techniques: (1) Genetic engineering — altering the chemistry of DNA/RNA to introduce into host organisms to change their phenotype. (2) Bioprocess engineering — maintaining sterile conditions in chemical engineering processes for large-scale production (bioreactor technology). Recombinant DNA technology was made possible by discovery of restriction endonucleases.
Tools of rDNA Technology
Restriction Endonucleases: Molecular scissors that cut DNA at specific recognition sequences (palindromic sequences). First discovered in E. coli by Werner Arber. EcoRI (from E. coli RY13): recognizes GAATTC, cuts between G and A on both strands → produces sticky ends (cohesive ends with overhanging bases). Some enzymes produce blunt ends. Sticky ends are useful for joining DNA from different sources using DNA ligase.
Cloning Vectors: Vehicles for carrying foreign DNA into host. Features needed: origin of replication (ori), selectable marker (antibiotic resistance gene), cloning sites (restriction sites), small size. Types: pBR322 (plasmid with ampicillin and tetracycline resistance genes, constructed by Bolivar and Rodriguez), pUC vectors, Bacteriophage lambda, BAC (Bacterial Artificial Chromosome), YAC (Yeast Artificial Chromosome). Insertional inactivation: Foreign DNA inserted into selectable marker gene inactivates it → recombinants identified. Blue-white screening: Insertion into lacZ gene → no beta-galactosidase → white colonies (recombinants); blue = non-recombinants.
Competent Host: Bacterial cells treated with CaCl2 (makes cell wall permeable) to take up DNA. Other methods: electroporation, gene gun (biolistics), microinjection.
Processes of rDNA Technology
Step 1: Isolation of DNA — Cell lysis using enzymes (lysozyme for bacteria, cellulase for plant cells), removal of RNA (RNase), proteins (protease), and purification. DNA precipitated with chilled ethanol (appears as fine threads/spooling).
Step 2: Cutting DNA — Restriction enzyme digests both foreign DNA and vector at same restriction site. Gel electrophoresis separates DNA fragments by size (smaller fragments move faster). DNA visualized under UV light after staining with ethidium bromide. Desired fragment cut out and extracted (elution).
Step 3: Ligation — Foreign DNA fragment joined to vector using DNA ligase → recombinant DNA molecule.
Step 4: Transfer to Host — Recombinant DNA introduced into competent host cell (transformation). Microinjection for animal cells, gene gun for plant cells.
Step 5: Screening — Identify transformants (cells with recombinant DNA) using selectable markers or blue-white screening.
Step 6: Scale-up — Desired recombinant grown in bioreactors (stirred-tank type is most common). Bioreactor provides optimal conditions: temperature, pH, aeration, agitation, substrate. Downstream processing: separation, purification, and formulation of product.
PCR (Polymerase Chain Reaction)
Invented by Kary Mullis. Amplifies specific DNA sequences in vitro. Requires: DNA template, two primers (forward and reverse), Taq polymerase (thermostable, from Thermus aquaticus), dNTPs. Three steps per cycle: Denaturation (94°C — strands separate), Annealing (primer binding, ~55-65°C), Extension (72°C — Taq polymerase synthesizes new strand). ~30 cycles → ~1 billion copies. Used in diagnostics, forensics, cloning.
Summary
Biotechnology uses genetic engineering and bioprocess engineering. Restriction enzymes cut DNA at specific sites. Vectors like plasmids carry foreign DNA into host cells. PCR amplifies DNA rapidly. rDNA technology involves DNA isolation, cutting, ligation, transformation, screening, and scale-up in bioreactors.
Important Terms
- Restriction enzyme: Enzyme cutting DNA at specific palindromic sequences
- Sticky ends: Overhanging single-stranded DNA ends produced by restriction enzymes
- Vector: DNA molecule that carries foreign DNA into host cell
- PCR: Polymerase Chain Reaction — in vitro DNA amplification
- Taq polymerase: Heat-stable DNA polymerase from Thermus aquaticus
- Transformation: Uptake of foreign DNA by competent host cell
- Bioreactor: Vessel for large-scale production of biotechnology products
- Palindrome: DNA sequence that reads same on both strands in 5'→3' direction
Quick Revision
- EcoRI: GAATTC recognition; produces sticky ends
- pBR322: AmpR and TetR markers; ori for replication
- Blue-white screening: lacZ gene; white = recombinant; blue = non-recombinant
- CaCl2 makes bacteria competent for DNA uptake
- PCR: Denaturation (94°C) → Annealing → Extension (72°C); uses Taq polymerase
- Gel electrophoresis: DNA moves to anode; smaller fragments travel farther
- DNA ligase joins DNA fragments (seals sugar-phosphate backbone)
- Gene gun/biolistics for plant cells; microinjection for animal cells