Biotechnology: Principles and Processes

This is the ninth chapter of NCERT's Class 12 Biology textbook (pages 163–176 of the 2026-27 reprint), the first of the two chapters of Unit IX, Biotechnology. It has three parts — principles of biotechnology (9.1), tools of recombinant DNA technology (9.2: restriction enzymes, cloning vectors, competent hosts) and processes of recombinant DNA technology (9.3: isolating and cutting DNA, PCR, insertion into the host, obtaining the product, downstream processing) — with Figures 9.1–9.7, a summary and 12 exercises. It is short but dense; this page uses eight concept sections.

• Neighbouring chapters: Molecular Basis of Inheritance (Chapter 5) supplies DNA structure, base pairing, replication and DNA polymerase, and uses these same tools in DNA fingerprinting; Microbes in Human Welfare (Chapter 8) introduced fermentors and streptokinase made by genetic engineering; Human Health and Disease (Chapter 7) met retroviruses such as HIV, which are disarmed here into vectors; Principles of Inheritance and Variation (Chapter 4) explains why breeding drags unwanted genes along. Chapter 10, Biotechnology and its Applications, follows with Bt cotton, insulin, gene therapy and the ethics debate.
• Prelims — science and technology: biotechnology defined (1993); genetic engineering to make a giant mouse (1993); restriction endonucleases and ligases cut and paste DNA (2001); who discovered endonucleases and PCR (2002); gene transfer across species by recombinant DNA (2013); DNA replicated outside a cell — PCR (2019); Cas9 as molecular scissors (2019); recombinant vector vaccines (2021); a clone as cells from one parent cell (1989).
• GS3 — Science and Technology: biotechnology, its tools and its uses in health and agriculture; diagnostics such as RT-PCR; biosafety regulation of genetically engineered organisms. GS2 touches it through health policy and drug regulation.

• Biotechnology = using live organisms or their enzymes for useful products and processes; today mainly with genetically modified organisms. EFB definition: integration of natural science and organisms, cells, parts thereof and molecular analogues for products and services. Two core techniques: genetic engineering and bioprocess engineering (sterile, large-scale growth).
• Principle: alien DNA multiplies only if linked to an origin of replication. Cohen and Boyer, 1972: an antibiotic-resistance gene cut from a plasmid and linked to a native plasmid of Salmonella typhimurium with DNA ligase → recombinant DNA → cloned in E. coli. Three steps: identify the gene, introduce it, maintain it and pass it on.
• Restriction enzymes (1963; first sequence-specific one Hind II, five years later; now 900+ from 230+ strains). EcoRI = Escherichia coli RY13, enzyme I. Endonucleases cut inside DNA, exonucleases nibble from the ends. They read palindromes (5′-GAATTC-3′ on both strands), cut between the same two bases a little off-centre → sticky ends → ligase joins.
• Gel electrophoresis: DNA (−) moves to the anode through agarose (from seaweed); smaller moves farther; ethidium bromide + UV → orange bands; cut out = elution. Vectors: ori (sets copy number), selectable marker (ampicillin, chloramphenicol, tetracycline, kanamycin resistance), single cloning sites; pBR322 — insert at BamHI in tetᴿ → insertional inactivation; β-galactosidase blue-white (white = recombinant); disarmed Ti plasmid of Agrobacterium tumefaciens, disarmed retroviruses.
• Getting DNA in: competent cells (divalent cation such as Ca²⁺), DNA on ice → 42 °C heat shock → ice; micro-injection (animal nucleus); biolistics/gene gun (gold or tungsten particles, plants); disarmed pathogens. Isolating DNA: lysozyme (bacteria), cellulase (plants), chitinase (fungi); RNase, protease; chilled ethanol precipitates threads → spooling.
• PCR: denaturation → primer annealing → extension, with two primers and a thermostable polymerase from Thermus aquaticus; 30 cycles ≈ 1 billion copies (2³⁰). Recombinant protein = expressed in a heterologous host. Continuous culture keeps cells in log phase. Bioreactors: 100–1000 L; stirred-tank (agitator, O₂ delivery, foam, temperature, pH control, sampling ports); sparged. Downstream processing: separation, purification, formulation with preservatives, clinical trials, quality control.

Biotechnology deals with techniques that use live organisms or their enzymes to make products and processes useful to humans. In that broad sense curd, bread and wine — all microbe-mediated — are biotechnology. Today the word is used more narrowly for processes that use genetically modified organisms to do this on a larger scale; it also covers in vitro fertilisation (a ‘test-tube’ baby), synthesising and using a gene, making a DNA vaccine and correcting a defective gene. The European Federation of Biotechnology (EFB) defines it, covering both old and new, as the integration of natural science and organisms, cells, parts thereof and molecular analogues for products and services.

Two core techniques gave birth to modern biotechnology. Genetic engineering alters the chemistry of genetic material (DNA and RNA), introduces it into a host and so changes the host's phenotype. Bioprocess engineering keeps a sterile (contamination-free) environment in chemical-engineering processes so that only the desired microbe or eukaryotic cell grows, in large quantities, to make antibiotics, vaccines, enzymes and the like. Why engineer at all? Sexual reproduction brings variation, but traditional hybridisation often carries undesirable genes along with the desired ones; recombinant DNA, gene cloning and gene transfer let us move only the gene or genes we want.

A piece of DNA dropped into an alien organism usually cannot multiply in the progeny cells. It is inherited only if it becomes part of something that replicates — a chromosome, which carries an origin of replication, the sequence where replication starts. So the alien DNA is linked to an origin of replication; it then replicates in the host. Making many identical copies of a template DNA this way is cloning.

The first recombinant DNA. In 1972 Stanley Cohen and Herbert Boyer cut an antibiotic-resistance gene out of a plasmid using a restriction enzyme — the ‘molecular scissors’ — and linked it to a native plasmid of Salmonella typhimurium (an autonomously replicating, circular, extra-chromosomal DNA). The plasmid acted as a vector, just as a mosquito is the vector that carries the malaria parasite. The joining was done by DNA ligase, which acts on cut DNA molecules and joins their ends. The result — circular, autonomously replicating DNA made in vitro — is recombinant DNA. Put into Escherichia coli, a close relative of Salmonella, it replicated using the host's DNA polymerase and made many copies: the cloning of the antibiotic-resistance gene in E. coli.

In 1963 two enzymes that restrict the growth of bacteriophages in Escherichia coli were isolated: one added methyl groups to DNA, the other cut DNA — the restriction endonuclease. Five years later the first restriction endonuclease whose action depended on a specific nucleotide sequence, Hind II, was isolated and characterised: it always cut DNA at a particular point by recognising a specific sequence of six base pairs, its recognition sequence. Today more than 900 restriction enzymes are known, from over 230 bacterial strains, each recognising a different sequence.

Naming. The first letter comes from the genus and the next two from the species of the prokaryote; then comes the strain; a Roman numeral gives the order in which enzymes were isolated from that strain. EcoRI = Escherichia coli strain RY13, enzyme I. Kinds of nucleases. Restriction enzymes belong to the nucleases: exonucleases remove nucleotides from the ends of DNA; endonucleases cut at specific positions within the DNA. Each restriction endonuclease ‘inspects’ the length of the DNA; on finding its recognition sequence it binds and cuts each of the two strands at specific points in their sugar-phosphate backbones.

Palindromes. A word palindrome reads the same forwards and backwards (MALAYALAM). A DNA palindrome is a stretch of base pairs that reads the same on the two strands when both are read in the same orientation: 5′-GAATTC-3′ on one strand, and 5′-GAATTC-3′ on the other (written below it as 3′-CTTAAG-5′). Every restriction endonuclease recognises a specific palindromic sequence. Restriction enzymes cut a little away from the centre of the palindrome but between the same two bases on both strands, leaving single-stranded overhangs — sticky ends, so called because they form hydrogen bonds with their complementary cut counterparts. This stickiness helps DNA ligase join them. DNA from any source cut by the same enzyme has the same sticky ends, so pieces can be joined end to end into recombinant DNA — which is why the vector and the source DNA must normally be cut with the same enzyme.

All eight sites read the same 5′→3′ on both strands (checked in the source file), so each is a palindrome — five of them answer NCERT exercise 7. Most leave sticky ends; PvuII and SmaI cut straight across and leave blunt ends, which ligase can still join, though less efficiently. NCERT's own example of a ‘six base pair’ enzyme, Hind II, is also a blunt cutter (Beyond NCERT).

Cutting DNA with restriction endonucleases gives fragments, which are separated by gel electrophoresis. DNA fragments are negatively charged, so in an electric field they are forced towards the anode (positive electrode) through a medium or matrix — today usually agarose, a natural polymer extracted from seaweeds. The gel acts as a sieve: fragments separate (resolve) by size, and the smaller the fragment, the farther it moves. DNA cannot be seen in visible light without staining, so the gel is stained with ethidium bromide and exposed to UV radiation, showing bright orange bands. A wanted band is cut out and the DNA extracted from the gel piece — elution. Fragments purified this way are joined with cloning vectors. Electrophoresis is also used to check the progress of a restriction digestion.

Plasmids and bacteriophages replicate inside bacteria independently of the chromosome. Phages, being numerous in a cell, have very high copy numbers; some plasmids have only one or two copies per cell, others 15–100, some more. Linking foreign DNA to them multiplies it to that copy number. Modern vectors are engineered for easy linking of foreign DNA and easy selection of recombinants from non-recombinants. Features needed:

• Origin of replication (ori) — where replication starts; any DNA linked to it replicates in the host. It also controls the copy number, so to recover many copies, clone in a vector whose ori supports a high copy number.
• Selectable marker — identifies and eliminates non-transformants and lets transformants grow. For E. coli the usual markers are genes for resistance to ampicillin, chloramphenicol, tetracycline or kanamycin; normal E. coli resists none of them. (Transformation = introducing a piece of DNA into a host bacterium.)
• Cloning sites — very few, preferably single, recognition sites for the common restriction enzymes. More than one site would cut the vector into several fragments and complicate cloning.
• Vectors for plants and animals — learnt from pathogens that already deliver genes into eukaryotic cells (below).

Insertional inactivation with two antibiotics. Foreign DNA is ligated at a site inside one of the two resistance genes — for example the BamHI site of the tetracycline-resistance gene in pBR322. Recombinant plasmids lose tetracycline resistance but keep ampicillin resistance. So the transformants are first plated on ampicillin (only cells with a plasmid grow), then transferred to tetracycline: recombinants grow on ampicillin but not on tetracycline; non-recombinants grow on both. One resistance gene selects transformants; the other, ‘inactivated due to insertion’, selects recombinants. This needs simultaneous plating on two plates and is cumbersome.

Blue-white selection. The alternative markers distinguish recombinants by colour in the presence of a chromogenic substrate. The insert is placed within the coding sequence of the enzyme β-galactosidase, inactivating its gene — insertional inactivation. Colonies whose plasmid has no insert make the enzyme and turn blue; colonies with an insert make no enzyme and stay colourless — these are the recombinant colonies.

Vectors for plants and animals. Agrobacterium tumefaciens, a pathogen of several dicot plants, delivers a piece of DNA called T-DNA that turns normal plant cells into a tumour making chemicals the pathogen needs. Its tumour-inducing (Ti) plasmid has been modified into a cloning vector that is no longer pathogenic but still delivers genes of our choice into many plants. Likewise, retroviruses, which can turn normal animal cells cancerous, have been disarmed to deliver desirable genes into animal cells. Once a gene is ligated into a suitable vector, it is transferred into a bacterial, plant or animal host, where it multiplies.

DNA is hydrophilic and cannot pass through cell membranes (its charged, water-loving backbone cannot cross the lipid layer). To force bacteria to take up a plasmid, the cells are made competent: treated with a specific concentration of a divalent cation such as calcium, which increases the efficiency with which DNA enters through pores in the cell wall. The recombinant DNA is then forced in by incubating the cells with it on ice, placing them briefly at 42 °C (heat shock), and putting them back on ice.

Other methods: micro-injection — recombinant DNA injected directly into the nucleus of an animal cell; biolistics or the gene gun — for plants, cells are bombarded with high-velocity micro-particles of gold or tungsten coated with DNA; and ‘disarmed pathogen’ vectors, which transfer the recombinant DNA when allowed to infect the cell.

Recombinant DNA technology runs in a fixed sequence: isolation of DNA, fragmentation by restriction endonucleases, isolation of the desired fragment, ligation into a vector, transfer of the recombinant DNA into the host, culturing the host at large scale, and extraction of the desired product.

Isolating DNA. DNA must be pure — free of other macromolecules — before it can be cut. Since it lies inside membranes, cells are broken open, releasing DNA with RNA, proteins, polysaccharides and lipids. Enzymes break the cell: lysozyme for bacteria, cellulase for plant cells, chitinase for fungi. Genes lie on long DNA molecules intertwined with proteins such as histones. Ribonuclease removes RNA, protease removes proteins, other treatments remove the rest, and purified DNA precipitates on adding chilled ethanol, visible as fine threads that can be spooled.

Cutting and joining. Purified DNA is incubated with the restriction enzyme under that enzyme's optimal conditions; agarose gel electrophoresis checks how the digestion is going (DNA moves to the anode). The vector DNA is cut the same way. The cut-out gene of interest and the cut vector ‘with space’ are mixed and ligase is added — giving recombinant DNA. Insertion into the host: competent recipient cells take up DNA from their surroundings. If the recombinant DNA carries, say, an ampicillin-resistance gene, transformed E. coli become ampicillin-resistant; spread on ampicillin agar, only transformants grow and untransformed cells die — the resistance gene is the selectable marker.

Obtaining the product. In almost every recombinant technology the aim is a desirable protein, so the recombinant DNA must be expressed, under appropriate conditions. A protein-encoding gene expressed in a heterologous (foreign) host gives a recombinant protein. Cells with the cloned gene can be grown on a small scale and the protein extracted and purified. They can also be grown in a continuous culture: used medium is drained out on one side while fresh medium is added on the other, keeping the cells in their physiologically most active log (exponential) phase — more biomass and higher yields.

PCR stands for Polymerase Chain Reaction. Multiple copies of a gene (or DNA) of interest are made in vitro using two sets of primers — small, chemically synthesised oligonucleotides complementary to regions of the DNA — and the enzyme DNA polymerase, which extends the primers using the nucleotides supplied and the genomic DNA as template. Each cycle has three steps: denaturation, primer annealing and extension. Repeated many times, the segment is amplified about a billion times. This is possible because the polymerase is thermostable — isolated from the bacterium Thermus aquaticus — and stays active through the high temperature used to denature the double-stranded DNA. The amplified fragment can then be ligated with a vector for cloning.

Small cultures cannot yield appreciable quantities, so bioreactors were developed, where 100–1000 litres of culture can be processed. They are vessels in which raw materials are biologically converted into specific products, individual enzymes and so on, using microbial, plant, animal or human cells. A bioreactor gives the optimum growth conditions — temperature, pH, substrate, salts, vitamins and oxygen. The most common are stirred-tank types: usually cylindrical or with a curved base to help mixing; the stirrer gives even mixing and oxygen throughout; alternatively, air can be bubbled through (a sparged stirred-tank reactor). A bioreactor has an agitator system, an oxygen delivery system, a foam control system, a temperature control system, a pH control system and sampling ports to withdraw small volumes periodically.

Downstream processing. After the biosynthetic stage, the product passes through separation and purification — together called downstream processing — before it can be marketed. It is formulated with suitable preservatives; the formulation, like any drug, undergoes thorough clinical trials; and strict quality-control testing is needed for each product. Both the processing and the testing vary from product to product.

The chapter asks six questions in its running text.

Numbers

One-liners

• Genetic engineering and bioprocess engineering are the two core techniques of modern biotechnology.
• Alien DNA multiplies only when linked to an origin of replication.
• Cohen and Boyer made the first recombinant DNA in 1972.
• Restriction enzymes are molecular scissors; DNA ligase is the glue.
• Endonucleases cut within DNA; exonucleases remove nucleotides from the ends.
• EcoRI comes from Escherichia coli strain RY13.
• Restriction enzymes recognise palindromic sequences.
• EcoRI cuts GAATTC between G and A, leaving sticky ends.
• Vector and source DNA must be cut with the same enzyme.
• DNA moves to the anode in gel electrophoresis; smaller fragments move farther.
• Agarose comes from seaweeds.
• Ethidium bromide and UV light make DNA bands visible.
• Cutting a band out of the gel and recovering DNA is elution.
• The ori controls copy number.
• pBR322 carries ampicillin- and tetracycline-resistance genes.
• An insert at BamHI in pBR322 inactivates tetracycline resistance.
• In blue-white screening, colourless colonies are recombinants.
• The Ti plasmid of Agrobacterium tumefaciens is disarmed into a plant vector.
• Calcium treatment and heat shock at 42 °C make bacteria take up DNA.
• The gene gun fires DNA-coated gold or tungsten particles into plant cells.
• Chilled ethanol precipitates purified DNA.
• Taq polymerase from Thermus aquaticus is heat-stable.
• PCR's steps are denaturation, annealing and extension.
• Continuous culture keeps cells in the log phase.
• Downstream processing means separation and purification of the product.

Traps to avoid

• In EcoRI, R stands for the strain RY13, not for restriction.
• Restriction enzymes cut off-centre, not at the centre of the palindrome.
• Ligase joins DNA; the restriction enzyme does not.
• DNA moves towards the anode, not the cathode.
• Smaller DNA fragments move farther, not slower.
• White colonies, not blue ones, carry the insert.
• Recombinants with a BamHI insert grow on ampicillin but not on tetracycline.
• Micro-injection is for animal cells; the gene gun is for plant cells.
• Lysozyme opens bacteria, cellulase plant cells, chitinase fungi.
• PCR copies double each cycle; they do not add.
• Taq polymerase matters because it is heat-stable.
• The first rDNA used a Salmonella plasmid but was cloned in E. coli.
• NCERT's 'tumifaciens' is spelt tumefaciens in standard sources.

Prelims — what UPSC asks

• Concepts and definitions: biotechnology as use of living organisms or their substances in industrial processes (1993); a mouse made giant with a human growth-hormone gene is genetic engineering (1993); a clone is a population of cells from one parent cell (1989); recombinant DNA moves genes across plant species, from animals to plants and from microbes to higher organisms (2013).
• Tools: restriction endonucleases and ligases let scientists cut and paste DNA from any source (2001); Arber and Smith — endonucleases, Feldman — Agrobacterium T-DNA transgenic plants, Mullis — PCR, Temin and Baltimore — reverse transcriptase (2002); Cas9 as molecular scissors for targeted gene editing (2019); DNA can be made to replicate outside a living cell — PCR (2019); genetic engineering and bacterial or viral vectors in recombinant vector vaccines (2021).
• Key notes: 2019 ‘which is not correct’ is keyed (a), that functional chromosomes can be made by joining DNA segments from cells of different species. Statements (b) artificial DNA, (c) DNA replication outside a cell (PCR) and (d) cell division in petri dishes are plainly true; (a) is the intended answer, though artificial chromosomes that carry foreign DNA do exist, so treat the key as debatable but standing. 1990 (effects of genetic engineering) and the hybridoma/monoclonal-antibody questions (2000, 2025) are not taught here.
• Likely next: naming of restriction enzymes, sticky vs blunt ends, the direction of DNA in a gel and what ethidium bromide does, features of a cloning vector and pBR322, insertional inactivation and blue-white screening, the Ti plasmid, competence and heat shock, the gene gun, the PCR steps and Taq, RT-PCR, CRISPR–Cas9, and the biosafety committees (IBSC, RCGM, GEAC).

Mains — where it appears and how to answer

• No Mains question asks about these tools alone; the biotechnology questions of GS3 (2018 on biopharma, 2019 on farmers, 2021 on applied biotechnology) are applications and belong with Chapter 10. This chapter supplies their technical spine: what recombinant DNA is, how a gene is cut, carried, copied and expressed, and how the product is scaled up and purified.
• Frame for any ‘biotechnology for development’ answer: tools (restriction enzymes, vectors, PCR, bioreactors) → products (recombinant medicines, vaccines, diagnostics, GM crops) → capacity (bioprocess engineering, downstream processing, quality control, clinical trials) → safeguards (Rules of 1989 under the Environment (Protection) Act, 1986; IBSC, RCGM, GEAC). Keep the GM-crop debate to attributed positions.

Tap an option. NCERT's own exercise questions come first; the rest test the figures and common confusions.

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