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Class 12 Biology

Biotechnology: Principles and Processes

Genetic engineering, restriction enzymes, vectors, competent hosts, steps of recombinant DNA technology, PCR and bioreactors — with SVG diagrams, an EcoRI digest simulator, MCQs, A-R and PYQs.

3NCERT Sections
12MCQs
6Assertion–Reason
5PYQs
Section 01

Principles of Biotechnology

9.1What is Biotechnology?

EFB definition

"The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services." Traditional processes (curd, bread, wine) and modern ones (IVF, gene synthesis, DNA vaccines, gene correction) are all biotechnology.

Two core techniques gave birth to modern biotechnology:

  • Genetic engineering: altering the chemistry of genetic material (DNA/RNA), introducing it into host organisms and changing the host's phenotype.
  • Bioprocess engineering: maintaining a sterile ambience so only the desired microbe/cell grows in large quantities for products like antibiotics, vaccines and enzymes.
Why genetic engineering?

Traditional hybridisation often includes and multiplies undesirable genes along with the desired ones. Genetic engineering (recombinant DNA, gene cloning, gene transfer) isolates and introduces only the desired gene(s).

9.1.1The First Recombinant DNA

  • Alien DNA multiplies only if linked to an origin of replication (ori) — the sequence that initiates replication. Making many identical copies = cloning.
  • Stanley Cohen and Herbert Boyer (1972) linked an antibiotic-resistance gene to a native plasmid of Salmonella typhimurium using restriction enzymes ('molecular scissors') and DNA ligase; the plasmid acted as a vector. Transferred into E. coli, it replicated using the host's DNA polymerase.
  • Three basic steps: (i) identification of DNA with desirable gene; (ii) introduction of this DNA into the host; (iii) maintenance of the introduced DNA in the host and its transfer to progeny.
V1 · Making recombinant DNA ① Cut with same enzyme source DNA (gene) + vector ② Sticky ends gene fragment + opened plasmid pair by H-bonds ③ DNA ligase RECOMBINANT DNA (joined in vitro) ④ Into host (E. coli) replicates with ori → multiple copies = cloning
Section 02

Tools of Recombinant DNA Technology

9.2Key Tools

Restriction enzymes, polymerase enzymes, ligases, vectors and the host organism.

9.2.1 Restriction Enzymes

  • In 1963 two enzymes restricting bacteriophage growth in E. coli were isolated: one added methyl groups to DNA, the other cut DNA — the restriction endonuclease.
  • The first: Hind II — always cuts at a specific 6 bp recognition sequence. Now >900 enzymes known from >230 bacterial strains.
  • Naming (EcoRI): E = genus Escherichia; co = species coli; R = strain RY 13; I = order of isolation.
  • Nucleases: exonucleases remove nucleotides from the ends; endonucleases cut at specific positions within DNA.
  • Each enzyme recognises a specific palindromic sequence (reads the same on both strands in 5′→3′): 5′ GAATTC 3′ / 3′ CTTAAG 5′. It cuts a little away from the centre, between the same two bases on opposite strands, leaving sticky (cohesive) ends that H-bond with complementary ends → easy action of DNA ligase.
  • Vector and source DNA must be cut with the same enzyme to give compatible ends.
V2 · EcoRI cuts G↓AATTC and leaves sticky ends BEFORE G A A T T C C T T A A G ··· ··· ··· ··· cut cut AFTER G C T T A A A A T T C G ··· ··· ··· ··· Sticky end — overhanging 5′ AATT Sticky end Palindrome: reads GAATTC on both strands (5′→3′)

V3Gel Electrophoresis

  • DNA fragments are negatively charged, so they move toward the anode (+) through a matrix — commonly agarose (polymer from sea weeds). Separation is by size (sieving): smaller fragments travel farther.
  • Bands are visible only after staining with ethidium bromide and exposure to UV — bright orange bands. Bands are cut out of the gel — elution — and used with vectors.
V3 · Agarose gel electrophoresis 1 2 3 4 5 (+) Anode (−) Cathode · wells Sample loaded in wells at the (−) end DNA is negatively charged → moves toward the anode (+) Small fragments move FARTHER (sieving effect of agarose) Ethidium bromide + UV → orange bands Lane 1 = undigested; others = digests
Section 02 (contd.)

Cloning Vectors

9.2.2Features of a Cloning Vector

Plasmids and bacteriophages replicate independently of chromosomal DNA. Plasmids have 1–2 copies or 15–100 copies per cell (bacteriophages have very high copy number). Linking alien DNA to them multiplies it equally.

Feature Role
Origin of replication (ori) Starts replication of linked DNA; controls copy number.
Selectable marker Identifies and eliminates non-transformants and lets transformants grow. Genes for resistance to ampicillin, chloramphenicol, tetracycline, kanamycin (normal E. coli is sensitive).
Cloning sites Very few, preferably single recognition sites for common enzymes; several sites would create many fragments and complicate cloning.
Vectors for plants/animals Ti plasmid of Agrobacterium tumefaciens (T-DNA; disarmed) for plants; disarmed retroviruses for animal cells.
V4 · pBR322 and selection by insertional inactivation ori amp R tet R rop BamH I Pst I pBR322 Insert DNA at the BamH I site inside tet R → tetracycline resistance is lost (insertional inactivation) Plate 1 — ampicillin: recombinants + non-recombinants both grow (transformants) Plate 2 — tetracycline: only NON-recombinants grow Grew on ampicillin but NOT on tetracycline = RECOMBINANT colonies Other sites: Hind III, EcoR I, Sal I, Pvu II, Pst I, Cla I · rop codes proteins for replication
Blue–white screening (alternative marker)

Foreign DNA is inserted within the coding sequence of β-galactosidase (insertional inactivation). With a chromogenic substrate: blue colonies = no insert (non-recombinant); colourless colonies = insert present (recombinant).

Section 02 (contd.)

Competent Host

9.2.3Getting DNA into the Host

DNA is hydrophilic and cannot cross cell membranes, so bacteria are made competent:

  • Divalent cation (calcium) treatment increases DNA entry through pores in the cell wall. Cells + recombinant DNA are kept on ice, given a brief heat shock at 42°C, then returned to ice.
  • Micro-injection — DNA injected directly into the nucleus of an animal cell.
  • Biolistics (gene gun) — plant cells bombarded with high-velocity gold or tungsten microparticles coated with DNA.
  • Disarmed pathogen vectors infect the cell and transfer the recombinant DNA.
Section 03

Processes of Recombinant DNA Technology

9.3Sequence of Steps

  1. Isolation of DNA → 2. Fragmentation with restriction endonucleases → 3. Isolation of desired fragment → 4. Ligation into a vector → 5. Transfer into host → 6. Culturing host cells at large scale → 7. Extraction of the product.

9.3.1 Isolation of the Genetic Material

  • DNA must be pure. Cells are broken with enzymes: lysozyme (bacteria), cellulase (plant cells), chitinase (fungi).
  • Remove RNA with ribonuclease, proteins with protease; other molecules by suitable treatments.
  • Purified DNA precipitates on adding chilled ethanol as fine threads that can be spooled.

9.3.2 Cutting and Joining

Purified DNA is incubated with the restriction enzyme under optimal conditions; digestion is checked by agarose gel electrophoresis. Vector DNA is cut the same way. The gene of interest and cut vector are mixed and ligase is added → recombinant DNA.

Try it · EcoRI digest simulator

Paste a DNA sequence (A, T, G, C). EcoRI recognises GAATTC and cuts between G and A.

Section 03 (contd.)

Amplification by PCR

9.3.3Polymerase Chain Reaction

PCR makes multiple copies of the gene/DNA of interest in vitro using two sets of primers (small chemically synthesised oligonucleotides complementary to regions of the DNA) and DNA polymerase. Repeated cycles amplify the segment about a billion times. A thermostable DNA polymerase from Thermus aquaticus (Taq polymerase) stays active during high-temperature denaturation.

V5 · One PCR cycle: denaturation → annealing → extension ① Denaturation high temperature separates the two DNA strands ② Primer annealing primers (purple) bind complementary regions ③ Extension Taq polymerase extends primers using dNTPs; template = DNA Repeat many cycles → ~1 billion copies · Taq polymerase ( Thermus aquaticus ) survives heat
  • The amplified fragment can then be ligated into a vector for further cloning.
  • Insertion into host: competent cells take up DNA; with an ampicillin-resistance marker, only transformants survive on ampicillin agar.
Section 03 (contd.)

Obtaining the Foreign Gene Product — Bioreactors

9.3.5Expression, Bioreactors and Downstream Processing

  • A gene expressed in a heterologous host gives a recombinant protein. Lab-scale cultures give little product; large quantities need bioreactors (100–1000 L).
  • Continuous culture: used medium drained from one side and fresh medium added from the other, keeping cells in the active log (exponential) phase → larger biomass and yield.
  • A bioreactor gives optimum temperature, pH, substrate, salts, vitamins and oxygen. The commonly used type is the stirred-tank reactor — a cylinder (often with curved base) where a stirrer mixes contents evenly and supplies oxygen; alternatively sterile air is sparged as bubbles. It has temperature and pH control systems and a sampling port.
  • Downstream processing — separation and purification of the product, formulation with suitable preservatives, clinical trials (for drugs) and quality control testing.
V6 · Stirred-tank bioreactor Motor Sterile air in sparged as bubbles (O₂) Stirrer (impeller) even mixing + O₂ supply Control systems temperature · pH Sampling / product outlet harvest for downstream Curved base → better mixing
Practice

📝 Solved Examples with Step Marking

📋 3 Marks

Q1. Why must the source DNA and vector be cut with the same restriction enzyme? What does DNA ligase do?

Same enzyme 1 Mark

The same enzyme leaves the same sticky ends on both DNAs.

Pairing 1 Mark

Complementary sticky ends form hydrogen bonds and pair end-to-end.

Ligase 1 Mark

DNA ligase joins the cut ends, producing recombinant DNA.

📋 3 Marks

Q2. Describe the three steps of PCR and name the enzyme used.

Denaturation 1 Mark

Heat separates the double-stranded DNA into single strands.

Annealing 1 Mark

Two sets of primers bind to complementary regions of the template.

Extension 1 Mark

Taq polymerase (from Thermus aquaticus) extends the primers using nucleotides — repeating the cycle gives ~10⁹ copies.

📋 3 Marks

Q3. How are recombinant colonies distinguished from non-recombinants by insertional inactivation of β-galactosidase?

Insertion 1 Mark

Foreign DNA is inserted into the coding sequence of the β-galactosidase gene, inactivating it.

Non-recombinants 1 Mark

Intact enzyme + chromogenic substrate → blue colonies.

Recombinants 1 Mark

No enzyme → colourless colonies.

Test Yourself

✍️ MCQ Practice

Score: 0 / 12
New Pattern

🎯 Assertion–Reason

(A) Both true, R explains A. (B) Both true, R does NOT explain A. (C) A true, R false. (D) A false, R true.

Score: 0 / 6
Exam Focus

📖 Important Board Questions

NCERT

Q. What are restriction enzymes? Explain palindromic sequence with an example.

Answer 3 Marks

Restriction endonucleases cut DNA at specific recognition sequences. A palindrome reads the same on both strands in the same 5′→3′ direction, e.g. 5′ GAATTC 3′ / 3′ CTTAAG 5′ (EcoRI). The cut leaves sticky ends.

NCERT

Q. List the features required of a cloning vector.

Answer 3 Marks

(i) Origin of replication (controls copy number); (ii) selectable marker (e.g. ampicillin/tetracycline resistance); (iii) unique cloning sites for restriction enzymes.

NCERT

Q. Explain PCR. Why is Taq polymerase used?

Answer 3 Marks

PCR amplifies DNA in vitro using primers and DNA polymerase through repeated denaturation, annealing and extension cycles (~10⁹ copies). Taq polymerase from Thermus aquaticus is thermostable and stays active at the high denaturation temperature.

NCERT

Q. How is a bacterial cell made competent to take up recombinant DNA?

Answer 2 Marks

Treated with a divalent cation such as calcium; then cells and DNA are kept on ice, given a heat shock at 42°C and returned to ice, so DNA enters the cell.

NCERT

Q. What is a bioreactor? Describe a stirred-tank bioreactor.

Answer 3 Marks

A vessel (100–1000 L) where raw materials are biologically converted to products under optimum temperature, pH, substrate, salts, vitamins and oxygen. A stirred-tank reactor has a cylindrical/curved-base tank, a stirrer for mixing and oxygen supply, a sparger for sterile air, and temperature/pH control.