Biotechnology: Principles and Processes Class 12 Notes जैव प्रौद्योगिकी : सिद्धांत व प्रक्रम — कक्षा 12 जीव विज्ञान अध्याय 9

Revise these first (prerequisites)

• Chapter 5: DNA structure, replication, 5'→3' direction
• Basic meaning of an enzyme (Class 11 Biomolecules)
• Plasmid = small circular DNA of bacteria (Chapter 8/Class 11 level)
• What antibiotics do (Chapter 8)

1. What is biotechnology?

   Using living organisms or their parts (enzymes, cells) to make products useful to humans — biotechnology (जैव प्रौद्योगिकी). Making curd is also an old form of biotechnology; in modern times, DNA is cut and joined to make useful products.

2. Genetic engineering = editing of DNA

   Taking a useful gene out of one organism and inserting it into another organism is genetic engineering. Think of DNA as a long sentence — we cut out one word (gene) and paste it into another sentence. This new DNA is called recombinant DNA (rDNA).

3. Scissors and glue

   The 'scissors' that cut DNA = restriction enzymes, which cut DNA only at a specific sequence. The 'glue' that joins the fragments = DNA ligase. Genetic engineering is not possible without both of these.

4. Vector = vehicle for DNA

   To carry a new gene into the host cell, a 'vehicle' is needed — this is called a vector, such as a bacterial plasmid. The vector replicates on its own inside the host, so many copies of the gene are also produced.

5. PCR = a photocopy machine for DNA

   PCR makes millions of copies of a small piece of DNA in the lab. Three steps are repeated again and again: heating to separate the strands (denaturation), binding of primers (annealing), and synthesis of the new strand (extension).

6. Large-scale product

   Once the rDNA-carrying bacterium is obtained, it is grown in large tanks — bioreactors — so that the product (such as insulin) is made in large quantities. The product is then separated and purified — downstream processing.

Quick check ✔

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As per NCERT 2026-27 — every point you need for the board exam.

1. 2 core techniques of biotechnology

   Genetic engineering (altering genetic material — rDNA) and bioprocess engineering (growing microbes/cells on a large scale under sterile conditions). EFB definition: integration of natural science and organisms/cells for products and services.

2. First rDNA

   Stanley Cohen & Herbert Boyer (1972): linked an antibiotic resistance gene into a plasmid of Salmonella typhimurium and introduced it into E. coli.

3. Restriction enzymes (molecular scissors)

   1963: two enzymes in E. coli. The first restriction endonuclease, Hind II, recognises a specific 6 bp sequence. Naming: EcoRI = E (genus Escherichia) co (coli) R (strain RY13) I (first). They cut at a palindromic sequence → sticky ends. Exonucleases cut from the ends, endonucleases cut within the DNA.

4. DNA ligase

   It joins cut DNA fragments together (molecular glue). Sticky ends make the work of ligase easier.

5. Gel electrophoresis

   DNA is negatively charged → moves towards the anode (+). Matrix: agarose (obtained from seaweed). Smaller fragments move farther. Ethidium bromide + UV light shows orange bands. Cutting out the band and extracting the DNA = elution.

6. Cloning vector

   Ori (starts replication, controls copy number), selectable marker (ampicillin, chloramphenicol, tetracycline, kanamycin resistance), cloning sites (a single recognition site). In pBR322, BamHI lies in the tetracycline gene; PvuI lies in the ampicillin gene.

7. Insertional inactivation

   Inserting the insert into the β-galactosidase gene. Recombinant colonies = colourless/white, non-recombinant = blue (in the presence of a chromogenic substrate). The ampicillin/tetracycline method is cumbersome, so this one is better.

8. Plant & animal vectors

   Agrobacterium tumefaciens — its Ti plasmid (tumour inducing) has been converted into a non-pathogenic vector. Retroviruses (in animals) are also disarmed and used as vectors.

9. Competent host

   DNA is hydrophilic and does not cross the cell membrane. Bacteria are treated with CaCl₂ (divalent Ca²⁺) → on ice → heat shock at 42°C → back on ice. Other methods: micro-injection (animal cell nucleus), biolistics / gene gun (plants — gold/tungsten particles), disarmed pathogen vectors.

10. Steps of rDNA technology

    Isolate DNA (lysozyme — bacteria, cellulase — plant, chitinase — fungus; RNase, protease; chilled ethanol precipitates fine threads of DNA) → restriction digestion → amplification by PCR → ligation into vector → transformation into host → culture for the product.

11. PCR

    Polymerase Chain Reaction: denaturation → annealing (primers) → extension. Uses thermostable Taq polymerase (Thermus aquaticus). Can produce ~1 billion copies.

12. Bioreactor & downstream

    Culture in large volumes (100–1000 L). Stirred-tank bioreactor: agitator, oxygen delivery, foam control, temperature and pH control, sampling ports. Continuous culture prolongs the log phase. Downstream processing: separation + purification, preservatives, clinical trials, quality control.

The concept in depth — the why and the how. Everything stays within the NCERT syllabus.

1. Why are sticky ends useful?

   Restriction enzymes recognise palindromic sequences and cut both strands a little away from the centre, leaving single-stranded overhangs — sticky ends. If the vector and source DNA are cut with the same restriction enzyme, their sticky ends are complementary, pair up by H-bonds, and DNA ligase joins them permanently. Hence using the same enzyme is the first requirement for making rDNA.

2. 3 must-haves of a vector

   ori: replication starts here and it controls copy number — the origin of replication from Chapter 5. Selectable marker (ampicillin, tetracycline, kanamycin resistance genes): distinguishes transformants from non-transformants. Cloning site: there should be only one recognition site for an enzyme, otherwise the vector will be cut into several pieces.

3. Insertional inactivation: a clever trick

   In pBR322, inserting foreign DNA at the BamHI site within tet^R causes loss of tetracycline resistance. So recombinant cells grow on ampicillin-containing medium but not on tetracycline. Replica plating on antibiotic plates is cumbersome, so now the insert is placed within the β-galactosidase gene and checked on a chromogenic substrate: non-recombinants are blue, recombinants are white (colourless).

4. Making the host 'competent'

   DNA is hydrophilic and cannot easily pass through the cell membrane. Bacteria are treated with a divalent cation (Ca²⁺), incubated with rDNA on ice, then given a heat shock at 42°C and placed back on ice — the DNA enters the cell. Other methods: micro-injection (animal cell nucleus), biolistics / gene gun (plants), and disarmed pathogen vectors such as the Ti plasmid of Agrobacterium.

5. The real hero of PCR: Taq polymerase

   Denaturation requires a high temperature, at which normal enzymes get destroyed. Taq polymerase, obtained from Thermus aquaticus, is thermostable, so fresh enzyme need not be added in every cycle. In this way, DNA can be amplified about 1 billion times — this is why, in Chapter 10, even very low amounts of a pathogen (HIV) can be detected by PCR.

Common confusions — cleared up

1. Exonuclease vs Endonuclease

   An exonuclease removes nucleotides from the ends of DNA; an endonuclease cuts at specific positions within the DNA.

2. Meaning of the name EcoRI

   E = genus Escherichia, co = species coli, R = strain RY 13, I = the order in which it was isolated from that strain.

3. Which way does DNA move in gel electrophoresis?

   DNA is negatively charged, so it moves towards the anode (+); smaller fragments move farther.

4. Transformant vs Recombinant

   Transformant = a cell that has taken up a plasmid (whether or not it carries rDNA). Recombinant = a cell that has taken up a plasmid carrying foreign DNA; insertional inactivation is used to identify these.

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