Biotechnology: Principles and Processes

NCERT starts by widening the word. Biotechnology, in the broad sense, is any use of living organisms or their enzymes to make products useful to us: making curd, bread, wine, even the biogas plant of the previous chapter. But the chapter is written about the narrow, modern meaning, and that is what the examiner tests. In the modern sense biotechnology means using genetically modified organisms or their products, and it covers in vitro fertilisation, gene synthesis, DNA vaccines, gene correction and similar work. The definition you should be able to quote is the one given by the European Federation of Biotechnology (EFB): biotechnology is 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. The first is genetic engineering: techniques that alter the chemistry of the genetic material (DNA or RNA) so that it can be introduced into a host organism and change the host's phenotype. The second is the maintenance of a sterile, microbial-contamination-free ambience in chemical engineering processes, so that only the desired microbe or eukaryotic cell grows in large quantities and makes the product we want, whether an antibiotic, a vaccine or an enzyme.

Why not simply cross organisms to bring in a new trait? Traditional hybridisation moves the desired gene along with a large number of undesirable genes, because whole genomes are mixed. Genetic engineering lets us isolate and introduce only one or a set of desirable genes. A piece of DNA put on its own into a cell cannot multiply, however; it is simply lost. It multiplies only when it becomes part of the host's chromosome, or when it is joined to a DNA that can replicate on its own, which means a DNA that carries an origin of replication (ori). Plasmids, the circular extra-chromosomal DNA of bacteria, replicate autonomously and so became the first carriers.

The first recombinant DNA was made in 1972 by Stanley Cohen and Herbert Boyer. They cut a gene for antibiotic resistance out of a plasmid of Salmonella typhimurium with a restriction enzyme, joined it to a native plasmid using DNA ligase, and transferred the new circular DNA into Escherichia coli, where it replicated using the host's DNA polymerase. Making many copies of a template this way is cloning. The process has three basic steps, which the examiner will ask you to list: (i) identification of DNA with the desirable gene, (ii) introduction of that DNA into the host, and (iii) maintenance of the introduced DNA in the host and its transfer to the progeny.

The key tools of recombinant DNA technology are restriction enzymes, polymerase enzymes, ligases, vectors and the host organism. The story of restriction enzymes begins in 1963, when two enzymes that restrict the growth of bacteriophage in E. coli were isolated. One added methyl groups to DNA (protecting the bacterium's own DNA), the other cut DNA. The cutting one was the restriction endonuclease. The first restriction endonuclease whose action depended on a specific DNA sequence, Hind II, was isolated and characterised five years later; it always cuts DNA at a particular point within a recognition sequence of six base pairs. Today more than 900 restriction enzymes have been isolated from over 230 strains of bacteria, each recognising a different sequence.

Naming convention (a favourite 3-mark question). Take EcoR I: E comes from the genus Escherichia, co from the species coli, R from the strain RY 13, and the Roman numeral I gives the order in which the enzyme was isolated from that strain. The first three letters are conventionally written in italics because they come from the organism's name.

Restriction enzymes belong to the larger class of nucleases. Exonucleases remove nucleotides from the ends of a DNA molecule; endonucleases cut at specific positions within the DNA. A restriction endonuclease works by 'inspecting' the length of a DNA molecule; once it finds its specific recognition sequence, it binds and cuts each of the two strands at specific points in their sugar–phosphate backbones. Each enzyme has its own recognition sequence, and that sequence is a palindrome.

A palindrome in language reads the same forwards and backwards ('MALAYALAM'). A palindrome in DNA is a sequence of base pairs that reads the same on the two strands when the orientation of reading is kept the same, that is, 5′→3′ on both. EcoR I recognises 5′-GAATTC-3′; the complementary strand read 5′→3′ is also GAATTC. Restriction enzymes cut a little away from the centre of the palindrome, between the same two bases on the opposite strands. EcoR I cuts between G and A on each strand, so each cut piece ends with a single-stranded overhang, AATT, called a sticky end. The ends are 'sticky' because they can form hydrogen bonds with a complementary sticky end, and this stickiness facilitates the action of DNA ligase. That is the whole reason the vector and the foreign DNA are cut with the same restriction enzyme: the two then carry matching sticky ends and can be joined end to end.

Cutting DNA with a restriction enzyme gives a mixture of fragments. To use them you must separate them, and the technique is gel electrophoresis. The principle rests on one fact: DNA fragments are negatively charged (because of the phosphate groups), so in an electric field they migrate towards the anode, the positive electrode. The migration is forced through a matrix, and the matrix most commonly used is agarose, a natural polymer extracted from sea weeds.

The agarose gel acts as a sieve. Fragments separate, or resolve, according to size through this sieving effect: the smaller the fragment, the farther it moves. So in a finished gel the smallest fragments are nearest the anode and the largest are nearest the wells. This is the sentence the marking scheme looks for when the question says 'state the basis on which fragments separate'.

The separated DNA is invisible. It is seen by staining the gel with ethidium bromide and then exposing it to UV radiation; the DNA appears as bright orange coloured bands. Pure DNA cannot be seen in visible light without staining, so a 1-mark question often reads 'DNA fragments in a gel can be visualised by' and the answer is ethidium bromide with UV light.

The band of interest is then cut out of the gel and the DNA is extracted from the gel piece. This step is called elution. The eluted, purified fragments are used to construct recombinant DNA by joining them to cloning vectors. A 5-mark question in recent papers asked for the whole sequence: principle, matrix, direction of movement, sieving, staining, visualisation and elution. Learn it as one paragraph and you can write it in any length the paper demands.

Two short points the examiner rewards. First, a lane loaded with a DNA of known fragment sizes (a marker or ladder) is run beside the samples so the size of an unknown band can be read off. Second, checking the progress of a restriction digestion is one of the everyday uses of agarose gel electrophoresis, which NCERT mentions again in section 9.3.2.

Plasmids and bacteriophages replicate inside bacterial cells independently of the chromosomal DNA, and that autonomy makes them useful as vectors: if a foreign DNA is linked to such a molecule, it multiplies along with it. Bacteriophages have very high copy numbers in a cell; some plasmids have only one or two copies per cell while others have 15 to 100 copies, and some even more. Many copies of the vector means many copies of the linked DNA, which is what cloning is about.

A vector is engineered to carry three features, and NCERT names them in this order.

(i) Origin of replication (ori). The sequence from which replication starts; any piece of DNA linked to it can be made to replicate inside the host. The ori also controls the copy number of the linked DNA. So if you want many copies of the target DNA, clone it into a vector whose ori supports a high copy number.

(ii) Selectable marker. A gene that helps in identifying and eliminating non-transformants and selectively permitting the growth of transformants. (Transformation is the procedure by which a piece of DNA is introduced into a host bacterium.) Normal E. coli cells carry no antibiotic resistance, so genes for resistance to ampicillin, chloramphenicol, tetracycline or kanamycin are useful markers: a cell that grows on the antibiotic must carry the vector.

(iii) Cloning sites. To link the alien DNA, the vector needs very few, preferably single, recognition sites for the commonly used restriction enzymes. More than one site for an enzyme would generate several fragments and complicate the cloning. The site should also lie inside a marker gene so that insertion can be detected (next section).

pBR322 is the vector everybody draws. It is a circular plasmid built for E. coli that carries an ori, two selectable markers, amp^R (ampicillin resistance) and tet^R (tetracycline resistance), the gene rop that codes for proteins involved in the replication of the plasmid, and unique restriction sites: Hind III, EcoR I, Cla I, BamH I, Sal I, Pvu II, Pst I and Pvu I. The sites BamH I and Sal I lie within tet^R; Pst I and Pvu I lie within amp^R; Pvu II lies near rop. In the 2025 paper several MCQs asked exactly this: which site to use for cloning into tetracycline resistance (Sal I or BamH I), into ampicillin resistance (Pst I or Pvu I), and near rop (Pvu II). The letters p, B and R in the name stand for plasmid and for Bolivar and Rodriguez, who constructed it; 322 is the laboratory number.

Insertional inactivation with antibiotic markers. Suppose the alien DNA is ligated at the BamH I site of the tetracycline-resistance gene of pBR322. The recombinant plasmid has lost tetracycline resistance because the gene is broken by the insert, but it still carries ampicillin resistance. Now plate the transformed E. coli on a medium with ampicillin: every cell carrying a plasmid, recombinant or not, grows; cells without a plasmid die. Then transfer the colonies onto a plate with tetracycline. Recombinants grow on ampicillin but not on tetracycline; non-recombinants grow on both. So one antibiotic selects transformants, and the second distinguishes recombinants from non-recombinants. The drawback, and NCERT says so, is that selection is cumbersome because it needs simultaneous plating on two plates with different antibiotics.

A better marker: β-galactosidase. The alternative is to insert the recombinant DNA inside the coding sequence of the enzyme β-galactosidase (the lacZ gene). If the plasmid has no insert, the gene is intact and, in the presence of a chromogenic substrate, the bacteria give blue coloured colonies. If the insert is present, β-galactosidase is inactivated by insertional inactivation and the colonies produce no colour: these white colonies are the recombinants. This is called blue–white selection, and it differentiates recombinants from non-recombinants on one plate, by colour. That single-step, single-plate identification is why the 2019 paper called it a 'better selectable marker' and asked you to justify it.

Vectors for cloning genes in plants and animals. Nature did the first experiments. Agrobacterium tumefaciens, a pathogen of several dicot plants, delivers a piece of DNA called T-DNA (from its Ti plasmid) into plant cells; the T-DNA transforms normal cells into a tumour and directs the tumour cells to produce chemicals the pathogen needs. Likewise retroviruses in animals transform normal cells into cancerous cells. Biotechnologists have disarmed both: the tumour-inducing (Ti) plasmid of Agrobacterium is modified so that it no longer causes disease but still delivers genes of interest into plant cells, and retroviruses are disarmed so that they deliver desirable genes into animal cells without causing cancer. That is why Agrobacterium is called a natural genetic engineer of plants.

Once a gene has been ligated into a suitable vector, the whole construct is transferred into the host: bacteria, plant cells or animal cells. The host must be ready to take DNA in, which is the next section.

DNA is a hydrophilic molecule, and hydrophilic molecules cannot pass through cell membranes. So a bacterium will not simply take up a plasmid from its surroundings; it has to be made competent, that is, capable of taking up DNA. This is done by treating the cells with a specific concentration of a divalent cation, such as calcium (Ca²⁺), which increases the efficiency with which DNA enters the bacterium through pores in its cell wall.

The recombinant DNA is then forced into such cells by a temperature routine you must be able to write in order: incubate the cells with the recombinant DNA on ice, then place them briefly at 42 °C (heat shock), then put them back on ice. The heat shock makes the bacteria take up the DNA. This calcium-chloride and heat-shock method is the standard way of transforming E. coli, and the 2024 five-mark question asked for exactly these points: why competence is needed, the calcium-ion treatment and the heat-shock sequence.

Since it is not always possible to get a cell to take up DNA this way, other methods deliver DNA directly into the host, and a 2-mark question routinely asks for two of them:

• Micro-injection: the recombinant DNA is directly injected into the nucleus of an animal cell with a fine needle.
• Biolistics or gene gun: cells, mostly plant cells, are bombarded with high-velocity micro-particles of gold or tungsten coated with DNA.
• Disarmed pathogen vectors: the pathogen (for example Agrobacterium) infects the cell and in doing so transfers the recombinant DNA into the host.

Keep the host examples straight because the paper does: micro-injection is for animal cells, the gene gun is for plant cells, calcium-heat shock is for bacteria, and Agrobacterium is for dicot plants. Whatever the route, once the DNA is inside, a vector with an ori replicates it and a selectable marker lets you find the cells that got it.

Section 9.3 walks through recombinant DNA technology as a sequence of steps, and NCERT lists them in one sentence you should memorise: isolation of DNA, fragmentation of DNA by restriction endonucleases, isolation of a desired DNA fragment, ligation of the DNA fragment into a vector, transferring the recombinant DNA into the host, culturing the host cells in a medium at large scale, and extraction of the desired product. The sections then take these one at a time.

To cut DNA with a restriction enzyme it must be in pure form, free from other macromolecules. DNA is enclosed within membranes, so the cell has to be opened first, and the enzyme used depends on the wall to be broken. This is one of the most reliable 1-mark questions in the paper:

• Lysozyme for bacteria.
• Cellulase for plant cells.
• Chitinase for fungi (including yeast).

Breaking the cell releases DNA along with RNA, proteins, polysaccharides and lipids, so the mixture is cleaned in stages. RNA is removed by treatment with ribonuclease; proteins are removed by treatment with protease; other molecules are removed by appropriate treatments. The 2019 paper asked why proteases are added, and the answer is that histones and other proteins bound to DNA and the enzymes released on lysis would otherwise contaminate, and possibly degrade, the DNA.

Finally the purified DNA is precipitated by the addition of chilled ethanol. Precipitated DNA can be seen as a collection of fine threads in the suspension, which can be spooled out on a glass rod. Every board answer on 'isolation of DNA' should contain these five words: lysozyme/cellulase/chitinase, ribonuclease, protease, chilled ethanol, fine threads.

Remember why the genome is such a difficult starting material: DNA molecules are very long and the gene of interest is a tiny fraction of them. Chapter 9 gives you two answers to that problem, cutting with restriction enzymes (next section) and amplifying by PCR (section 9.3.3).

Restriction enzyme digestion is done by incubating the purified DNA with the chosen restriction enzyme under the optimal conditions of temperature and pH for that enzyme. The progress of the digestion is checked by agarose gel electrophoresis: an uncut plasmid, a linearised plasmid and a mixture of fragments all run differently, so a small sample of the reaction on a gel tells you whether the cut is complete. Since DNA is negatively charged, it moves towards the positive electrode, and the fragments resolve by size as described in section 9.2.1.

The same procedure is done on the vector DNA, with the same restriction enzyme. That point matters. Because both the source DNA and the vector are cut with one enzyme, every end produced carries the same sticky end, and a fragment of the source DNA can pair by base complementarity with the opened vector. The joining of the cut-out gene of interest to the cut vector is done with DNA ligase, which forms phosphodiester bonds between the sugar–phosphate backbones. The product is recombinant DNA.

NCERT's flow diagram of recombinant DNA technology is a 3-mark question in itself, and it appeared as a fill-the-blanks figure in the 2025 paper. Reproduce it in words: a bacterial plasmid vector and a foreign DNA containing the gene of interest are both cut with the same restriction enzyme, producing sticky ends; the pieces are mixed and joined by DNA ligase to give a recombinant plasmid; the recombinant plasmid is introduced into a bacterial host by transformation; the host multiplies, and because the vector carries an ori the recombinant DNA is copied along with it; a selectable marker lets you pick out the transformed cells.

A separation step sits between cutting and ligation. After digestion, the mixture contains many fragments, and only the one carrying the gene is wanted. It is separated by gel electrophoresis, the band is eluted, and then the pure fragment is ligated to the cut vector. Some boards' answers describe the gene of interest being 'isolated' before ligation; they mean this elution step.

Two terminology cautions. DNA ligase joins the two DNA molecules covalently; the sticky ends only hold them together temporarily by hydrogen bonds until ligase acts. And the bond formed by ligase is a phosphodiester bond between adjacent nucleotides on the same strand, not a hydrogen bond and not a glycosidic bond, a distinction the 2026 Assertion–Reason item tested.

PCR stands for Polymerase Chain Reaction. It makes multiple copies of a gene (or any segment of DNA) in vitro, using two sets of primers and the enzyme DNA polymerase. Primers are small, chemically synthesised oligonucleotides that are complementary to the regions of DNA flanking the segment to be copied; one primer binds each strand. The enzyme extends the primers using the nucleotides (dNTPs) provided in the reaction and the genomic DNA as template. Each cycle of the reaction has three steps, and the 5-mark answer needs all three with their purpose:

1. Denaturation. The double-stranded DNA is heated to a high temperature (about 94 °C) so that the hydrogen bonds break and the two strands separate. Each strand can now act as a template.
2. Annealing. The temperature is lowered (typically to 50–65 °C, depending on the primers) so that the two primers base-pair with their complementary sequences on the two separated strands, one at each end of the target segment.
3. Extension (polymerisation). The temperature is raised to the optimum of the polymerase (about 72 °C) and the enzyme extends each primer in the 5′→3′ direction, adding nucleotides complementary to the template.

After one cycle there are two copies of the segment; after two cycles four; after n cycles about 2ⁿ. Repeated over about 30 cycles this amplifies the segment approximately a billion times, that is, about 1 billion copies from a single template.

The enzyme cannot be ordinary DNA polymerase, because the denaturation step would destroy it every cycle. So PCR uses a thermostable DNA polymerase isolated from the bacterium Thermus aquaticus, called Taq polymerase, which remains active during the high-temperature denaturation of the double-stranded DNA. This is the answer to 'why a thermostable polymerase, and from where', asked for 2 marks in 2025 and for 5 marks in 2017 and 2019 as 'describe the roles of heat, primers and the bacterium Thermus aquaticus in PCR'.

The amplified fragment can then be ligated into a vector for further cloning, which is where PCR joins the recombinant DNA workflow. In Chapter 10 you will meet PCR again as a diagnostic tool: it can detect a pathogen's DNA or a mutated gene when the amount of DNA is far too small to see by other means. When drawing the PCR figure, show the template being denatured, the two primers annealing at opposite ends on opposite strands, and Taq polymerase extending each primer, repeated for the second cycle to show four molecules.

There are several ways to introduce the ligated DNA into recipient cells, and section 9.2.3 covered them: calcium-chloride treatment and heat shock for bacteria, micro-injection for animal cells, the gene gun for plant cells, and disarmed pathogens like Agrobacterium. Whichever route is used, the recipient cells are first made competent, that is, able to take up the DNA present in their surroundings.

The important new idea in this section is how you know the DNA went in. NCERT's example: if a recombinant DNA bearing the gene for ampicillin resistance is transferred into E. coli cells, the host cells become transformed into ampicillin-resistant cells. If the transformed cells are spread on agar plates containing ampicillin, only the transformants grow; untransformed cells die. In this case, since the ampicillin-resistance gene was used to pick out the cells that received the DNA, it is being used as a selectable marker.

Read that carefully, because the paper asks it as an Assertion–Reason item: the antibiotic in the plate is not there to kill contaminants, it is the selection itself. A gene that lets a cell survive under a condition that kills all others is a selectable marker; the ampicillin plate is the selection condition.

Two small but examinable distinctions follow. A transformant is any cell that has taken up the vector; a recombinant is a transformant whose vector carries the insert. Selecting transformants needs one marker (grow on ampicillin); distinguishing recombinants from non-recombinants needs a second signal, either a second antibiotic gene inactivated by the insert or the β-galactosidase colour test. In an exam you will sometimes be given a plate diagram and asked which colonies to pick; the recombinant colonies are the ones that grow on ampicillin but not on tetracycline, or the white ones on a blue–white plate.

Once the transformed host has been selected, it can be grown up. The host's own replication machinery copies the vector, and its transcription and translation machinery can express the gene, which takes us to the product.

The purpose of cloning a gene is usually the protein it codes for. After cloning, the gene has to be expressed, that is, transcribed and translated in the host, and the protein it makes is called a recombinant protein. Cells carrying the cloned gene are grown on a small scale in the laboratory and the culture is used to extract the protein and purify it. But small-volume cultures cannot yield appreciable quantities of product, so to produce in large amounts, bioreactors were developed. A bioreactor is a vessel in which large volumes (100 to 1000 litres) of culture can be processed; raw materials are biologically converted into specific products, individual enzymes and so on, using microbial, plant, animal or human cells.

A bioreactor provides the optimal conditions for achieving the desired product: optimum temperature, pH, substrate, salts, vitamins and oxygen. The most commonly used bioreactors are of the stirring type. A stirred-tank reactor is usually cylindrical or has a curved base to facilitate the mixing of the contents. The stirrer facilitates even mixing and oxygen availability throughout the reactor. Alternatively, air can be bubbled through the reactor; a reactor with such an air inlet (a sparger) is the sparged stirred-tank bioreactor, and it is the one you should be able to draw and label.

List the components as NCERT does, because the 3-mark question is often 'name the components and their functions': an agitator (stirrer) system, an oxygen delivery system, a foam control system, a temperature control system, a pH control system, and sampling ports so that small volumes of the culture can be withdrawn periodically for testing.

Bioreactors can be run in batch mode, where the medium is loaded, the culture grows and everything is harvested, or as a continuous culture system. In the continuous system, the used medium is drained out from one side while fresh medium is added from the other, keeping the cells in their physiologically most active log/exponential phase. This gives a larger biomass and a higher yield of the product, and 'how and why is a continuous culture maintained' was a 3-mark question in 2019.

The NCERT exercise also asks what advantages a stirred-tank bioreactor has over a shake flask besides aeration and mixing. Write: large volume, precise and automatic control of temperature and pH, controlled oxygen delivery, foam control, aseptic operation, and sampling ports for monitoring without disturbing the culture.

Growing the culture is the upstream half of the job. After the biosynthetic stage is complete, the product has to be taken through a series of processes before it is ready for marketing as a finished product. These processes are collectively called downstream processing, and they consist of two main things: separation of the product from the culture (cells, medium and debris) and purification of the product to the required standard.

The purified product then has to be formulated with suitable preservatives so that it stays stable during storage. In the case of drugs, the formulation has to undergo thorough clinical trials before it can be used on patients, exactly as any new medicine must. And strict quality control testing is required for each product, batch by batch.

NCERT ends the chapter with one sentence you should keep: the downstream processing and quality control testing vary from product to product. An enzyme for a detergent, an insulin for injection and a vaccine for a child are separated, purified, formulated and tested to very different standards. That variation is the reason the section is short in the book and yet turns up in the 5-mark bioreactor question as 'what happens after the product is made'.

For a 2-mark answer, write four steps in order: separation of the product from the culture, purification, formulation with preservatives, and quality control (with clinical trials for drugs). For the flow-of-the-chapter question, downstream processing is the last box after 'culturing the host cells in a medium at large scale'. In Chapter 10 the recombinant insulin story picks up from here: the A and B chains made in E. coli are extracted, purified and combined, and that combination is the downstream step for that product.

A final check on vocabulary that separates full marks from partial marks in this chapter: 'transformation' is introducing DNA into a host; 'cloning' is making many copies; 'expression' is making the protein; 'downstream processing' is everything after the culture. Use each word for its own step and the answer reads like the marking scheme.