Biotechnology: Principles and Processes

Written and reviewed by Anjali Sharma, Biology faculty at UniDrill (M.Sc. Botany, Banaras Hindu University · 8 years teaching NCERT Biology). Based on NCERT Biology Class XII, Chapter 9.

Can you cut DNA with a pair of scissors?

You cannot, but bacteria have been doing it for millions of years. Biotechnologists borrowed their molecular scissors, their DNA glue and their self-copying rings of DNA, and learned to move a gene from one organism to another. That is how the insulin in a diabetic's syringe is made by bacteria. This chapter is the toolbox: what each tool is, why it works and the order in which it is used.

What is biotechnology?

Biotechnology is the use of living organisms, cells or enzymes to make useful products and processes. The European Federation of Biotechnology (EFB) defines it as the integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services. In the narrower modern sense, it means using genetically modified organisms at large scale.

Two core techniques made modern biotechnology possible.

Why a gene alone is not enough. If you push a piece of foreign DNA into a bacterium, it will usually just sit there and be lost as the cell divides. To be copied, a DNA piece needs an origin of replication (ori), the sequence from where replication starts. So the foreign DNA must be linked to a carrier that has an ori. Once it is replicated along with the host, you can make many copies of it, which is cloning: producing multiple identical copies of any template DNA.

The founding experiment. In 1972, Stanley Cohen and Herbert Boyer built the first artificial recombinant DNA. They cut an antibiotic-resistance gene out of one plasmid with restriction enzymes and joined it to a native plasmid of Salmonella typhimurium using DNA ligase. They then put it into E. coli, where it replicated. The idea was simple: if you can cut and join DNA, you can make any combination you want.

The three steps of genetic modification: (i) identify the DNA carrying the desired gene, (ii) introduce it into the host, (iii) maintain it in the host and pass it to the progeny.

The toolbox: five things you need

To do rDNA technology you need restriction enzymes (scissors), DNA ligase (glue), polymerases (copiers), vectors (carriers) and a host organism (the factory).

Restriction enzymes: the molecular scissors

Bacteria use restriction enzymes to defend themselves against invading viruses. In 1963, scientists found two enzymes in E. coli that stop the growth of bacteriophages: one adds methyl groups to DNA and the other cuts it. The cutting enzyme is the restriction endonuclease. A bacterium protects its own DNA by methylating it, and chops up the unmethylated DNA of an intruder. The first restriction endonuclease to be characterised, Hind II, came in 1968. It was found to cut DNA at a specific sequence of six base pairs, the recognition sequence. More than 900 restriction enzymes have now been isolated from over 230 bacterial strains.

Two kinds of nucleases. Exonucleases remove nucleotides from the ends of DNA. Endonucleases cut at specific places inside the DNA. A restriction enzyme is an endonuclease.

How the name is built. Take EcoRI: E is the genus (Escherichia), co is the species (coli), R is the strain (RY 13), and I is the order of isolation (the first enzyme from that strain). The letter R is the strain, not "restriction".

Palindromes and sticky ends. A restriction enzyme recognises a palindromic sequence, a stretch of base pairs that reads the same on both strands when you read each in the same 5' to 3' direction. For EcoRI it is 5'-GAATTC-3' on one strand and 3'-CTTAAG-5' on the other. The enzyme cuts both strands, but not at the centre: it cuts between G and A on each strand. Because the cuts are in different places on the two strands, short single-stranded overhangs are left behind. These are sticky ends. They hydrogen-bond with any complementary sticky end cut by the same enzyme, and that makes it easy for DNA ligase to seal the joint.

The principle in one line: cut the vector and the foreign DNA with the same enzyme → matching sticky ends → they pair → DNA ligase seals the nicks → recombinant DNA.

Pause and predict: the sequence 5'-GGATCC-3' is a palindrome in the DNA sense. What is its complementary strand read in the 5' to 3' direction?

Check your answer: also 5'-GGATCC-3'. That is the whole definition: the sequence reads the same on both strands when each is read 5' to 3'. A word palindrome like "madam" reads the same backwards. A DNA palindrome reads the same on the other strand.

Beyond the textbook. For the exam: restriction enzymes cut DNA at palindromic recognition sites and leave sticky ends. In reality: not every restriction enzyme leaves sticky ends. Some cut straight across the centre and leave blunt ends, which can also be joined, but less efficiently. Why it matters: the exam says "sticky ends", but if a question says "blunt", the answer changes.

Separating DNA: gel electrophoresis

After cutting, you have a mixture of fragments of different sizes. Gel electrophoresis sorts them out.

DNA is negatively charged because of its phosphate backbone, so in an electric field it moves towards the positive electrode, the anode. The fragments are pushed through a gel made of agarose, a natural polymer extracted from seaweeds. The gel acts like a sieve, so smaller fragments travel farther. After the run, the DNA is stained with ethidium bromide and seen as bright orange bands under UV light. The band you want is cut out of the gel and the DNA is recovered. This step is called elution.

The steps in order: DNA migrates towards the anode through the agarose gel → staining with ethidium bromide → exposure to UV light → bands seen → elution of the band.

Beyond the textbook. For the exam: DNA is stained with ethidium bromide and seen under UV light. In reality: ethidium bromide slips between the base pairs of DNA, and that is why it glows and also why it is a mutagen and must be handled carefully. Many laboratories now use safer dyes. Why it matters: the dye is the reason a stained gel needs careful handling, and UV is used only to make the stained bands visible.

Vectors: the carriers

A vector is a piece of DNA that carries the foreign gene into the host and copies it. A good cloning vector has four features.

pBR322, the classic E. coli vector, is a plasmid. The p stands for plasmid, B and R for its makers (Bolivar and Rodriguez), and 322 is a number for that plasmid. It carries an ori, a gene called rop (coding for proteins needed for plasmid replication), two antibiotic-resistance genes, ampR (ampicillin) and tetR (tetracycline), and unique recognition sites for Hind III, EcoR I, BamH I, Sal I, Pvu II, Pst I and Cla I.

How the antibiotic trick picks out recombinants. This is a favourite question, so work it through. The BamH I site lies inside the tetR gene.

This is insertional inactivation: the foreign DNA interrupts a gene and switches it off. You first plate on ampicillin to keep every transformant, then transfer to tetracycline. The colonies that grew on the first plate but die on the second are the recombinants.

A second marker: colour. Insertional inactivation of the gene for the enzyme β-galactosidase gives the same information by colour. On a plate with a chromogenic substrate, colonies with a functional enzyme turn blue (no insert) and those with the enzyme inactivated by the insert stay colourless (recombinant). This is simpler than antibiotics because only one plate is needed.

Vectors for plants and animals. The Ti plasmid of Agrobacterium tumefaciens, a bacterium that naturally causes tumours (crown gall) in plants, is made harmless (disarmed) and used to deliver genes into dicot plants. Disarmed retroviruses do the same for animal cells.

Beyond the textbook. For the exam: the Ti plasmid is a vector for dicot plants. In reality: Agrobacterium does not infect most monocots in nature, but with changed protocols scientists now transform monocots such as rice and maize with it too. Why it matters: "dicots" describes the natural host range of the bacterium, and the rule is a starting point, not a law.

Getting the DNA into the host

DNA is a large, water-loving (hydrophilic) molecule, so it cannot just slide through a cell membrane. The host must be made ready to accept it.

Transformation, in order: treat with calcium → incubate with rDNA on ice → heat shock at 42 °C → back on ice → plate on antibiotic agar to select transformants.

Pause and predict: why must the Ca2+ treatment come before the rDNA is added, not after?

Check your answer: the calcium is what makes the cell competent. Without it the rDNA has no easy way in, so the bacteria have to be prepared first.

The process of rDNA technology, step by step

The sequence below is the path CUET expects, from the cell to the finished product.

Step 1 in detail. The cell wall has to come off, and the enzyme depends on the cell type.

After that, ribonuclease removes RNA and protease removes proteins. When chilled ethanol is added, pure DNA separates as a fine, thread-like precipitate that can be lifted out by spooling.

PCR: photocopying DNA

PCR (Polymerase Chain Reaction) makes millions of copies of a chosen gene in a test tube. You need the template DNA, two primers, the enzyme and nucleotides. Primers are short, chemically synthesised oligonucleotides complementary to the ends of the target region. Each cycle has three steps.

The enzyme used is Taq polymerase, from the bacterium Thermus aquaticus, which lives in hot springs. Its great advantage is that it is thermostable: it survives the high temperature of denaturation. An ordinary DNA polymerase, such as that of E. coli, would be destroyed in the first cycle. After about 30 cycles the gene is amplified roughly a billion times, because the number of copies doubles every cycle.

Beyond the textbook. For the exam: PCR has three steps, denaturation, annealing and extension, and Taq polymerase is thermostable. In reality: PCR was conceived by Kary Mullis, who shared the Nobel Prize in Chemistry in 1993. Taq polymerase has no proofreading ability, so a copy now and then has an error, and laboratories that need very accurate copies use other thermostable polymerases. The same method, in a form that starts from RNA (RT-PCR), was widely used to test for the COVID-19 virus. Why it matters: the temperatures and the doubling rule are what the exam builds questions on, and the real-world uses show why the technique matters.

Making the product in bulk

When a gene is expressed in a host of a different kind, the protein made is called a recombinant protein. A small culture in the lab can make only a little. Cells can also be grown in a continuous culture system, where used medium is drained from one side and fresh medium is added from the other. This keeps the cells in their most active log (exponential) phase and gives a larger biomass and a higher yield.

For larger quantities, scientists use bioreactors, vessels in which 100 to 1000 litres of culture can be processed. A bioreactor gives the best conditions for growth, with the right temperature, pH, substrate, salts, vitamins and oxygen.

The most common design is the stirred-tank bioreactor, a cylindrical vessel (often with a curved base) to improve mixing. In a sparged stirred-tank bioreactor, sterile air is released as bubbles, which greatly increases the area available for oxygen transfer.

Downstream processing. After production, the product is not ready to use. It must be separated and purified, formulated with suitable preservatives, and put through clinical trials (for drugs) and strict quality-control testing. This varies from product to product.

Exam traps

60-second revision

Where this chapter connects

• Earlier: Molecular Basis of Inheritance explains DNA structure, replication and the central dogma that all these tools depend on.
• Next: Biotechnology and its Applications shows what these tools are used for, from Bt cotton to insulin and gene therapy.
• Microbes link: Microbes in Human Welfare covers the industrial fermentors that bioreactors grew out of.