Recombinant DNA Technology: Tools, PCR, DNA Fingerprinting and CRISPR

Biotechnology and Genetic Engineering: Principles

What Is Biotechnology: Traditional and Modern

Biotechnology is old in practice and new in method. Making curd, bread or wine uses microbes, so in a broad sense it is biotechnology. Today the word is used in a narrower sense, for processes that use genetically modified organisms to make products on a large scale. In vitro fertilisation, making a gene and using it, developing a DNA vaccine and correcting a defective gene all fall within it.

The European Federation of Biotechnology gives a definition that covers both the old and the new: ‘the integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services’. Two techniques made modern biotechnology possible:

• Genetic engineering: Techniques that alter the chemistry of the genetic material, DNA and RNA, introduce it into a host organism and so change the host’s characteristics.
• Bioprocess engineering: Keeping a large culture free of contaminating microbes, so that only the desired microbe or cell grows, in the quantities needed to make antibiotics, vaccines and enzymes.

Genetic Engineering and Recombinant DNA: Why One Gene Can Be Moved

Traditional breeding mixes two whole sets of genes, so undesirable genes often travel along with the one that is wanted. Genetic engineering avoids this. Through recombinant DNA, DNA joined from different sources, gene cloning and gene transfer, it isolates one desirable gene, or a set of them, and introduces only those into the target organism.

A piece of foreign DNA put into a cell usually cannot multiply on its own. It multiplies only when it is linked to a DNA sequence called the origin of replication, the point where copying starts. Linked to such a sequence, the foreign DNA is copied along with the host's own DNA and passed to the next generation. Making many identical copies of a piece of DNA in this way is called cloning.

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, joined it to another plasmid, and put the result into Escherichia coli, which copied it. Every genetic modification still follows the three steps they used:

1. Identify the DNA that carries the desirable gene.
2. Introduce that DNA into the host.
3. Keep the introduced DNA in the host and pass it on to the host’s progeny.

Tools of Recombinant DNA Technology

Restriction Enzymes: The Molecular Scissors

Recombinant DNA technology needs a small set of tools: restriction enzymes to cut DNA, polymerase enzymes to copy it, ligases to join it, vectors to carry it and a host organism to keep it. Of these, the restriction enzymes came first. In 1963 two enzymes that restrict the growth of viruses in E. coli were isolated; one added methyl groups to DNA and the other cut it.

The first restriction enzyme whose cut depended on a particular DNA sequence, Hind II, was described five years later. It always cut DNA at a sequence of six base pairs, its recognition sequence. More than 900 restriction enzymes are now known, isolated from over 230 strains of bacteria. Werner Arber, Hamilton Smith and Daniel Nathans shared the Nobel Prize in Physiology or Medicine in 1978 for discovering this class of enzymes.

• Naming restriction enzymes: The first letter comes from the genus and the next two from the species of the bacterium, then the strain and a Roman number for the order of discovery. EcoRI comes from Escherichia coli RY 13.
• Exonucleases and endonucleases: Exonucleases remove nucleotides from the ends of DNA; endonucleases, which include the restriction enzymes, cut at specific positions within it.
• Palindromic sites: Each restriction enzyme recognises a palindrome, a stretch that reads the same on both strands in the same direction, such as GAATTC paired with CTTAAG.
• Sticky ends: Most of these enzymes cut a little away from the centre of the site, between the same two bases on each strand. This leaves short single-stranded overhangs that pair with any other end cut by the same enzyme, which makes joining easy. EcoRI, for example, cuts GAATTC between the G and the A on each strand and leaves four-base AATT overhangs.

DNA ligase is the enzyme that then seals the joint. Because a vector and a gene cut by the same restriction enzyme carry matching sticky ends, ligase can join them into one recombinant molecule; cut with different enzymes, they usually cannot be joined.

Gel Electrophoresis: Separating DNA Fragments

Cutting DNA gives a mixture of fragments, and gel electrophoresis sorts them by size. DNA carries a negative charge, so in an electric field it moves through a gel towards the positive electrode, the anode. The usual gel is agarose, a natural polymer extracted from seaweed, which acts as a sieve: the smaller the fragment, the farther it moves.

• Seeing the bands: DNA is invisible in ordinary light. It is stained with ethidium bromide and seen as bright orange bands under ultraviolet light.
• Elution: A band is cut out of the gel and the DNA is extracted from it, then used to build recombinant DNA.
• Checking a digest: The same method shows whether a restriction enzyme has finished cutting.

Gene Cloning Vector: Plasmids, pBR322 and Ti Plasmid

A cloning vector is the DNA molecule that carries a foreign gene into a host and multiplies it there. Plasmids, small circles of DNA that copy themselves inside bacteria, and bacteriophages, viruses of bacteria, are the usual vectors. A plasmid may exist in one or two copies per cell or in 15 to 100, and a linked gene is multiplied to the same number. A useful vector has four features:

• Origin of replication (ori): The sequence where copying starts; it also sets the copy number of the linked DNA.
• Selectable marker: A gene, usually for resistance to an antibiotic such as ampicillin, tetracycline or kanamycin, that lets only transformed cells survive.
• Cloning sites: Preferably a single recognition site for each common restriction enzyme, so the vector is opened at one place only.
• A way to spot recombinants: In pBR322, an E. coli vector, a gene inserted at the BamH I site inactivates tetracycline resistance; recombinant cells grow on ampicillin but not on tetracycline. In newer vectors the insert breaks the gene for β-galactosidase, so recombinant colonies stay colourless while the others turn blue, a method called insertional inactivation.

Genes are moved into plants and animals with tools borrowed from pathogens. Agrobacterium tumefaciens naturally transfers a piece of DNA, the T-DNA, into the cells of dicot plants and turns them into tumours. Its tumour inducing (Ti) plasmid has been disarmed into a vector that no longer causes disease but still delivers genes. Retroviruses, which can turn animal cells cancerous, have been disarmed in the same way to deliver genes into animal cells.

Competent Host and Methods of Gene Transfer

DNA cannot pass through a cell membrane on its own, so the host cell has to be made competent to take it up. Bacteria are treated with a divalent cation such as calcium, incubated with the recombinant DNA on ice, given a brief heat shock at 42 °C and returned to ice. Other hosts need other methods:

• Microinjection: Recombinant DNA is injected directly into the nucleus of an animal cell.
• Biolistics or gene gun: Plant cells are bombarded with tiny particles of gold or tungsten coated with DNA.
• Disarmed pathogens: A vector such as the disarmed Ti plasmid or a retrovirus infects the cell and carries the DNA in.

Process of Recombinant DNA Technology

Recombinant DNA Technology Steps: From Gene to Product

1. Isolation of DNA: Cells are broken open with enzymes, lysozyme for bacteria, cellulase for plant cells and chitinase for fungi. Ribonuclease removes RNA and protease removes proteins, and chilled ethanol makes the pure DNA come out as fine threads.
2. Cutting at specific locations: The DNA and the vector are cut with the same restriction enzyme, and agarose gel electrophoresis checks the cut.
3. Joining: The gene of interest and the opened vector are mixed with DNA ligase, which forms the recombinant DNA.
4. Amplification by PCR: Where many copies of the gene are needed first, they are made by the polymerase chain reaction.
5. Insertion into the host: Competent cells take up the recombinant DNA, and a selectable marker such as ampicillin resistance picks out the transformed cells.
6. Obtaining the product: The host expresses the gene. A protein made from a gene expressed in a foreign host is called a recombinant protein.

PCR Technique: The Polymerase Chain Reaction

The polymerase chain reaction (PCR) makes a very large number of copies of one stretch of DNA in a test tube. It needs the DNA to be copied, two primers, short pieces of DNA made chemically to match the two ends of the stretch, the four nucleotides and a DNA polymerase. The American biochemist Kary Mullis invented it in 1983.

1. Denaturation: The mixture is heated to 94 to 98 °C, and the two strands of the DNA separate.
2. Annealing: It is cooled to 50 to 65 °C, and the primers bind to their matching places on each single strand.
3. Extension: At about 72 °C the polymerase extends the primers into new strands.

Each cycle doubles the stretch between the primers, so repeated cycles amplify it to about a billion copies. This works because the enzyme used, Taq polymerase from the heat-loving bacterium Thermus aquaticus, survives the high temperature of every denaturation step. Before it came into use, fresh enzyme had to be added after every cycle.

Bioreactors and Downstream Processing

A gene that works in a laboratory flask has to be scaled up to give useful amounts of product. Cells can be kept in their most active phase by continuous culture, in which used medium is drained from one side while fresh medium is added from the other. For large quantities, the culture is grown in a bioreactor, a vessel that holds 100 to 1,000 litres and keeps temperature, pH, nutrients and oxygen at their best.

• Stirred-tank bioreactor: The most common type, a cylinder with a curved base; a stirrer, or sterile air bubbled through it, mixes the culture and spreads oxygen.
• Control systems: An agitator, an oxygen supply, foam control, temperature and pH control, and ports for taking small samples.
• Downstream processing: Separation and purification of the product, its formulation with preservatives, clinical trials for a drug and strict quality control.

DNA Fingerprinting and Genome Sequencing

What Is DNA Fingerprinting: VNTR and Uses

About 99.9 per cent of the bases in human DNA are the same in all people, so comparing whole genomes is slow. DNA fingerprinting looks only at repetitive DNA, regions where a short sequence is repeated many times. These regions are part of the satellite DNA, classed as microsatellites, minisatellites and so on; they do not code for proteins, and the number of repeats varies greatly from person to person.

Alec Jeffreys at the University of Leicester developed the technique in 1984. He used a minisatellite called the variable number of tandem repeats (VNTR), whose length varies from 0.1 to 20 kilobases. The original method had six steps:

1. Isolation of DNA.
2. Digestion with restriction enzymes.
3. Separation of the fragments by electrophoresis.
4. Blotting of the fragments onto a nitrocellulose or nylon membrane (Southern blotting).
5. Hybridisation with a labelled VNTR probe.
6. Detection of the bands by autoradiography.

The bands form a pattern particular to each person. DNA from blood, hair follicles, skin, bone, saliva or sperm gives the same pattern, which makes it a tool of forensic science, and the repeats are inherited from parents, which makes it the basis of paternity testing. Since PCR, DNA from a single cell is enough. Laboratories today mostly compare short tandem repeats (STRs), the shorter repeats of microsatellites.

Microsatellite DNA, DNA Barcoding and Environmental DNA

The same variation that identifies a person also separates populations and species. Microsatellites, short sequences repeated in tandem, mutate faster than most DNA, so they vary a great deal. They are used in DNA profiling and kinship analysis. In population genetics they measure how closely subspecies and populations are related, which makes them a tool for studying evolutionary relationships.

• DNA barcoding: A short, standard stretch of DNA identifies a species, much as a barcode identifies a product. For animals the usual region is part of the mitochondrial gene cytochrome c oxidase I (COI). It tells apart species that look alike and finds undeclared plant or animal material in processed food; it cannot tell the age of an organism.
• Environmental DNA (eDNA): Organisms shed DNA into soil, water and air. Collected and sequenced, it shows which species live in a habitat, including endangered animals that are seldom seen.
• Metagenomics: The study of all the genetic material from all the organisms in an environment, without culturing each species. Aerial metagenomics collects the DNA carried in the air of a habitat in one go.

Human Genome Project and Genome Sequencing

Reading the full sequence of bases in an organism's DNA is called genome sequencing. Frederick Sanger's method of 1977 was the most widely used for about 40 years; massively parallel or next-generation sequencing, which reads a huge number of DNA fragments at the same time, has since cut the cost greatly. The yeast Saccharomyces cerevisiae was the first eukaryote to have its genome completely sequenced, released in 1996.

The Human Genome Project began in 1990 and was completed in 2003, a 13-year effort coordinated by the US Department of Energy and the National Institutes of Health, with partners in Britain, Japan, France, Germany and China. The genome was broken into fragments, cloned in bacterial and yeast artificial chromosomes, sequenced and reassembled by computer, a task that gave rise to bioinformatics, the use of computers to store and analyse biological data. Its main findings:

• The human genome has 3,164.7 million bases.
• Less than 2 per cent of it codes for proteins; repeated sequences make up a very large part.
• Chromosome 1 has the most genes, 2,968, and the Y chromosome the fewest, 231.
• About 1.4 million places where single bases differ between people, the single nucleotide polymorphisms (SNPs), were found; they help locate genes linked to disease.

Gene Maps, Crop Genomes and the Genome India Project

A map of where genes and DNA markers sit on the chromosomes has practical uses. Markers mapped in cattle, pigs and chickens are used to detect parentage, and a marker linked to disease resistance lets breeders pick resistant plants and animals without waiting to see the disease, a method called marker-assisted selection.

Sequencing a crop does the same work on a larger scale. In 2011-12 Indian scientists decoded the genome of pigeonpea, the first plant genome sequenced entirely by Indian institutions; of its 47,004 protein-coding genes, 1,213 are for disease resistance and 152 for tolerance to drought, heat and salinity, and the sequence is expected to speed up the breeding of new varieties. Reading a pathogen's genome shows the effector proteins it uses to attack a crop, which helps breeders find the crop's matching resistance genes. The full set of RNA molecules expressed in a cell, the transcriptome, shows which genes are actually in use.

• Genome India: The Department of Biotechnology sequenced the whole genomes of 10,074 healthy individuals from 83 populations at 99 sites, about 36.7 per cent rural, 32.2 per cent urban and 31.1 per cent tribal.
• Indian Biological Data Centre: The data, released on 9 January 2025, is stored at this national repository and shared with researchers under the Biotech-PRIDE Guidelines; a future target of sequencing 10 million genomes has been announced.
• Why an Indian dataset: A catalogue of Indian genetic variation allows diagnostic chips, diagnostics and therapies designed for India’s own populations.

Genome Editing and CRISPR-Cas9

CRISPR Full Form and How CRISPR-Cas9 Works

Genome editing changes the DNA of a living organism at a chosen place, instead of adding a gene from outside as older genetic engineering did. Its best-known tool began as a bacterial defence. CRISPR sequences in bacteria and archaea store pieces of the DNA of viruses that once attacked them; the Spanish microbiologist Francisco Mojica was among the first to study these repeats. When the same virus returns, an RNA copied from the stored piece guides the protein Cas9 to the virus's DNA, and Cas9 cuts it.

1. Find: A guide RNA carries a 20-nucleotide sequence that matches the target. Cas9 checks the DNA and binds only where the match lies next to a short protospacer adjacent motif (PAM), such as NGG.
2. Cut: Cas9, an RNA-guided endonuclease, cuts both strands, a double-strand break.
3. Repair by joining the ends: The cell’s non-homologous end joining (NHEJ) seals the break but often adds or deletes a few bases, which knocks the gene out.
4. Repair from a template: If a piece of DNA with the desired change is supplied, homology-directed repair (HDR) copies it, giving a precise edit.

In 2012 Jennifer Doudna and Emmanuelle Charpentier fused the two natural RNAs into a single guide RNA, turning the system into a tool that can be aimed at almost any gene by changing that guide alone. They received the Nobel Prize in Chemistry in 2020. Newer forms cut less and edit more finely: base editing changes a single nucleotide without breaking the DNA strands, and prime editing, a ‘search-and-replace’ method, writes new genetic information directly into the target site.

CRISPR Gene Editing: Uses, Ethics and Regulation in India

Genome editing is used in crops, in medicine and in research. In agriculture it can change a plant's own genes without leaving foreign DNA behind, which is why it is regulated differently from older transgenic crops. In medicine it can repair the gene behind an inherited disease in a patient's own cells. Editing such body cells, somatic editing, affects only the patient; editing eggs, sperm or embryos, germline editing, passes the change to future generations.

• Rules of 1989: Work with genetically engineered organisms in India falls under the Rules for the manufacture, use, import, export and storage of hazardous microorganisms and genetically engineered organisms or cells, 1989, notified under the Environment (Protection) Act, 1986, which the Ministry of Environment, Forest and Climate Change administers.
• Laboratory oversight: Laboratory work is overseen by Institutional Biosafety Committees (IBSCs) and the Review Committee on Genetic Manipulation (RCGM).
• Genome-edited plants: An office memorandum of 30 March 2022 exempted genome-edited plants of the SDN-1 and SDN-2 categories that carry no foreign DNA from Rules 7 to 11, and standard operating procedures for their review followed the same year. In May 2025 India announced its first two genome-edited rice varieties, DRR Rice 100 (Kamala) and Pusa DST Rice 1.
• Therapies: Gene-editing therapies are approved by the Central Drugs Standard Control Organisation under the New Drugs and Clinical Trials Rules, 2019, guided by the National Guidelines for Gene Therapy Product Development and Clinical Trials, 2019, and the ICMR’s ethical guidelines. Stem cell guidelines restrict editing of germ-line cells and human embryos to in vitro studies.

Recombinant DNA Technology Uses and Applied Biotechnology in India

Recombinant DNA Technology Uses: Medicine, Farming and Industry

The applications of biotechnology include therapeutics, diagnostics, genetically modified crops, processed food, bioremediation, waste treatment and energy production. Three research areas decide how well any of them works: the best catalyst, which is an improved microbe or a pure enzyme; the best conditions for that catalyst; and downstream processing to purify the product.

Medicine is the largest of these fields, and the article on gene therapy and biotechnology in medicine covers it in detail: insulin, gene therapy, molecular diagnosis and biopharmaceuticals. Vaccines and the immune system are explained in the article on immunity and vaccines, and stem cells and cloning in the article on the cell cycle and stem cells.

Biotechnology in India: DBT, BIRAC and the BioE3 Policy

India set up a separate Department of Biotechnology (DBT) in 1986, under the Ministry of Science and Technology, to administer the development and commercialisation of biotechnology. Its institutes include the Centre for DNA Fingerprinting and Diagnostics in Hyderabad, and its public sector undertaking, the Biotechnology Industry Research Assistance Council (BIRAC), funds start-ups through schemes such as the Biotechnology Ignition Grant.

• BioE3 Policy: Biotechnology for Economy, Environment and Employment, approved by the Union Cabinet on 24 August 2024, aims to make India a hub of high-performance biomanufacturing in chemicals, enzymes, agriculture and therapeutics, through biomanufacturing hubs, Bio-AI hubs and biofoundries.
• Size of the bioeconomy: India’s bioeconomy grew from 10 billion dollars in 2014 to 165.7 billion dollars in 2024, and the government targets 300 billion dollars by 2030.
• Start-ups: Biotechnology start-ups grew from about 50 in 2014 to over 8,500 in 2023.

These tools reach the poor in concrete ways. Tests based on PCR detect HIV and other infections early; Bt crops protect themselves from insects and need less chemical pesticide; genome-edited rice is bred for yield and for saving water; and indigenous gene therapies, such as the CAR-T therapy NexCAR19 and BIRSA 101 for sickle cell disease, cost a fraction of the treatments sold abroad.

Sources

• NCERT: Biology (Class XII), Biotechnology: Principles and Processes
• NCERT: Biology (Class XII), Biotechnology and its Applications
• NCERT: Biology (Class XII, earlier edition), Molecular Basis of Inheritance
• PIB: Genome India data released through the Indian Biological Data Centre (9 January 2025)
• PIB: Parliament question, Indian Biological Data Centre (19 March 2025)
• PIB: Exemption of SDN-1 and SDN-2 genome-edited plants (2022)
• PIB: Standard operating procedures for genome-edited plants under SDN-1 and SDN-2 (27 October 2022)
• PIB: Two genome-edited rice varieties announced (4 May 2025)
• PIB: BIRSA 101, India's first indigenous CRISPR-based gene therapy (19 November 2025)
• PIB: Parliament question, self-reliance in CRISPR and gene editing (19 March 2026)
• PIB: Parliament question, BioE3 Policy
• PIB: The rise of India's bioeconomy (27 March 2025)
• PIB: India's first home-grown CAR-T cell therapy (4 April 2024)
• ICAR: DARE/ICAR Annual Report 2011-12, Crop Improvement
• ICMR and DBT: National Guidelines for Stem Cell Research, 2017
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• Wikipedia: Cas9
• Wikipedia: CRISPR gene editing
• Wikipedia: Genome editing
• Wikipedia: Polymerase chain reaction
• Wikipedia: Restriction enzyme
• Wikipedia: Recombinant DNA
• Wikipedia: DNA profiling
• Wikipedia: STR analysis
• Wikipedia: Microsatellite
• Wikipedia: DNA barcoding
• Wikipedia: Environmental DNA
• Wikipedia: Metagenomics
• Wikipedia: Transcriptome
• Wikipedia: Massive parallel sequencing
• Wikipedia: Sanger sequencing
• Wikipedia: Saccharomyces cerevisiae
• Wikipedia: Taq polymerase
• Wikipedia: Hamilton O. Smith
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