Biotechnology
1. Introduction
Biotechnology uses living organisms or their components to develop useful products. Modern biotechnology relies on genetic engineering techniques to manipulate DNA.
1.1 History and Milestones
| Year | Milestone | Scientist/Organisation |
|---|---|---|
| 1972 | First recombinant DNA molecule | Paul Berg |
| 1973 | First plasmid cloning vector | Cohen and Boyer |
| 1978 | First human insulin gene expressed in E. coli | Genentech |
| 1982 | Humulin approved for human use | Eli Lilly |
| 1983 | First transgenic plant (tobacco) | Zambryski et al. |
| 1990 | Human Genome Project begins | International consortium |
| 1996 | Dolly the sheep cloned | Ian Wilmut |
| 2003 | Human genome sequence completed | HGP Consortium |
2. Principles of Biotechnology
2.1 Genetic Engineering
Direct manipulation of an organism's genome using recombinant DNA technology. Involves:
- Identification of a gene of interest.
- Insertion into a vector.
- Introduction into a host organism.
- Expression of the gene.
2.2 Core Techniques
- Restriction enzymes: Cut DNA at specific recognition sites (palindromic sequences).
- DNA ligase: Joins DNA fragments (seals the sticky ends).
- Vectors: Vehicles to carry foreign DNA (plasmids, bacteriophages, YACs, BACs).
- PCR: Amplifies specific DNA sequences.
- Gel electrophoresis: Separates DNA fragments by size.
3. Tools of Biotechnology
3.1 Restriction Enzymes
Recognize palindromic sequences (4-8 bp). Cut to produce sticky ends or blunt ends.
Example: EcoRI cuts GAATTC between G and A.
3.2 Cloning Vectors
Plasmid: Small, circular extrachromosomal DNA. Features:
- Origin of replication (ori): Replication control.
- Selectable marker: Antibiotic resistance (ampR, tetR).
- Cloning site: Multiple cloning site (MCS) with unique restriction sites.
Bacteriophage: Lambda phage, M13 phage.
BAC and YAC: For large DNA inserts (used in genome sequencing).
3.3 Polymerase Chain Reaction (PCR)
Amplifies a specific DNA region exponentially. Uses:
- DNA template
- Primers (two, forward and reverse)
- Taq polymerase (thermostable, from Thermus aquaticus)
- dNTPs
- Buffer with Mg²⁺
Cycles: Denaturation (95°C) → Annealing (50-65°C) → Extension (72°C). 30 cycles give ~1 billion copies.
4. Applications of Biotechnology
4.1 Bt Cotton (Insect Resistance)
Bt toxin gene from Bacillus thuringiensis inserted into cotton. Produces cry proteins that kill bollworms.
4.2 Insulin Production (Humulin)
Human insulin gene inserted into E. coli. Produces proinsulin, which is processed into active insulin. First recombinant DNA therapeutic approved for human use (1982).
4.3 Gene Therapy
Correction of genetic disorders by introducing functional genes. Example: SCID (severe combined immunodeficiency) treated with ADA gene therapy.
4.4 GMOs
Genetically modified organisms: Bt brinjal, Golden Rice (β-carotene enriched), Flavr Savr tomato.
5. Ethical Issues
- Safety of GMOs for human consumption and environment.
- Patenting of genes and organisms.
- Gene editing (CRISPR) in human embryos.
- Biopiracy and benefit sharing.
6. Worked Problems
Problem 1: A plasmid has a single EcoRI site. Foreign DNA cut with EcoRI is inserted. How can recombinants be identified? Solution: Use insertional inactivation of a marker gene (e.g., lacZ). Recombinants are white (blue-white screening).
7. Common Mistakes
'Students often confuse the roles of restriction enzymes and DNA ligase. Restriction enzymes cut DNA. DNA ligase joins DNA fragments.'
8. ISC Exam Focus
| Topic | Theory Marks | Practical Marks |
|---|---|---|
| Tools (enzymes, vectors, PCR) | 4 | 2 |
| Applications (Bt, insulin) | 4 | 2 |
| Gene therapy | 2 | 1 |
| Ethical issues | 2 | 1 |
9. Self-Test Questions
- Describe the structure and features of a cloning vector.
- Explain the principle and steps of PCR. Why is Taq polymerase used?
- How is genetically engineered insulin produced? Why is it better than animal-derived insulin?
- What are the advantages and concerns of genetically modified crops?
- Write a short note on the use of restriction enzymes in recombinant DNA technology.
