Biotechnology — Principles and Applications
Why This Chapter Matters
Biotechnology gives 10-12 marks in CBSE Class 12. Recombinant DNA technology, PCR, gel electrophoresis, cloning vectors, and applications (insulin, GMO, gene therapy) are all tested.
Core Concepts
1. Tools of Biotechnology
Restriction Enzymes (Molecular Scissors):
Cut DNA at specific palindromic sequences.
Type II restriction enzymes used in rDNA technology.
Produce blunt ends (e.g., Sma I) or sticky ends (cohesive ends with overhangs e.g., EcoR I, Hind III).
EcoR I cuts at 5'-GAATTC-3' producing 4-base overhangs.
Named from organism: E=Escherichia, co=coli, R=strain, I=first enzyme.
DNA Ligase (Molecular Glue): Joins sticky ends. Creates phosphodiester bond.
Vectors: Carry foreign DNA into host.
Types: Plasmids (circular bacterial DNA), Bacteriophages, Cosmids, BAC, YAC.
Properties required: Origin of Replication (ori), Selectable Marker (antibiotic resistance), Cloning Site (restriction enzyme sites — MCS).
pBR322: first artificial plasmid. Has ampicillin (amp^R) and tetracycline (tet^R) resistance genes.
2. Recombinant DNA Technology (rDNA)
Steps:
Transformation methods:
Heat shock (bacteria), Electroporation, Biolistics (gene gun — for plants), Microinjection.
Selectable markers: Insert foreign DNA into antibiotic resistance gene → insertional inactivation.
Example: insert into tet^R gene of pBR322 → cells with insert are amp^R but tet^S.
3. PCR (Polymerase Chain Reaction)
Amplifies specific DNA sequence in vitro.
Components: Template DNA, Primers (2 short ssDNA flanking target), Taq polymerase (heat-stable, from Thermus aquaticus), dNTPs, buffer.
Cycle (3 steps, 30-35 cycles):
After n cycles: 2ⁿ copies of target sequence.
Uses: DNA fingerprinting, diagnosis, forensics, paternity testing, detecting HIV.
4. Gel Electrophoresis
Separates DNA fragments by size.
DNA = negatively charged → moves toward positive electrode (+) in electric field.
Agarose gel: smaller fragments move faster, farther.
Ethidium bromide staining → bands visible under UV light.
5. Cloning Vectors — Details
Ti plasmid: From Agrobacterium tumefaciens. Used to introduce genes into plants. T-DNA integrates into plant chromosome.
Retroviruses: Used to deliver genes into animal cells (gene therapy).
6. Applications
Insulin production:
Human insulin gene (A and B chain) inserted into E.coli plasmids.
Proinsulin produced → chains extracted and combined with correct disulphide bonds.
humulin = recombinant human insulin. Replaced pig/cow insulin.
Bt toxin (Bacillus thuringiensis):
cry genes encode insecticidal proteins (Bt toxins).
Bt cotton: cry1Ac and cry2Ab genes → protects against bollworm.
Bt brinjal: cry1Ac → resistant to fruit borer.
Golden Rice: Contains β-carotene synthesis genes (daffodil + soil bacterium). Provides Vitamin A precursor to prevent deficiency.
Gene Therapy:
Replacing defective gene with functional copy.
First approved: ADA (Adenosine Deaminase) deficiency in children.
Challenges: immune response, insertion mutations, transient expression.
DNA Fingerprinting: Based on Variable Number of Tandem Repeats (VNTRs). Used in forensics, paternity, identification.
PYQs (CBSE)
CBSE 2023: What is insertional inactivation? How is it used to screen recombinants?
When foreign DNA is inserted into a restriction site WITHIN an antibiotic resistance gene (e.g., tet^R in pBR322), the gene is inactivated. Non-recombinants: amp^R AND tet^R. Recombinants: amp^R but tet^S (can't grow on tetracycline). Screening by replica plating on both media.
CBSE 2022: Explain PCR. What is the role of Taq polymerase?
PCR amplifies specific DNA in vitro using 3 temperature cycles: denaturation(94°C), annealing(50-65°C), extension(72°C). Taq polymerase (from Thermus aquaticus) is heat-stable and works at 72°C — it synthesises new DNA strands from the primers. After n cycles: 2ⁿ copies.