What is biotechnology and genetic engineering?
Biotechnology means using living things or their parts (cells, enzymes) to make useful products. Making curd from milk is old biotechnology. Modern biotechnology rests on two ideas:
- Genetic engineering: changing the genetic material (DNA or RNA) of a living thing, so its phenotype changes.
- Keeping things sterile (aseptic conditions): so that only the wanted microbe or cell grows, not germs from outside.
Why not just use normal breeding? Breeding mixes thousands of genes at once, including unwanted ones. Genetic engineering moves only the one gene we want. The first recombinant DNA was made in 1972 by Stanley Cohen and Herbert Boyer: they joined an antibiotic-resistance gene into a plasmid of Salmonella typhimurium.
Why is a vector needed? A piece of DNA put alone into a cell usually cannot copy itself. It must join a DNA that has an origin of replication (ori), the place where copying starts. A plasmid has an ori, so the gene copies along with it.
Tools of recombinant DNA technology
1. Restriction enzymes (molecular scissors)
These enzymes cut DNA at a fixed sequence called the recognition site. The first ones found were in bacteria, where they cut the DNA of attacking viruses. They belong to a larger group called nucleases: exonucleases remove letters from the ends; endonucleases cut inside the DNA. Restriction enzymes are endonucleases. The recognition site is a palindrome: read 5′→3′ it is the same on both strands. Example EcoRI site: 5′-GAATTC-3′ and on the other strand 3′-CTTAAG-5′ (read backwards = GAATTC). Many enzymes cut a little away from the centre, leaving sticky ends that pair easily with any DNA cut by the same enzyme.
Naming rule: EcoRI = E (genus Escherichia) + co (species coli) + R (strain RY13) + I (first enzyme found in that strain). More than 900 restriction enzymes are known. (Hind II was the first, found in 1970s; it cuts at a 6-letter site too.)
2. DNA ligase (molecular glue)
Joins the sugar-phosphate backbone of the gene and the vector.
3. Cloning vectors
Plasmids and bacteriophages. A good vector needs:
- ori: starts copying and decides the copy number.
- Selectable marker: a gene (like resistance to ampicillin, tetracycline, kanamycin) that lets us pick transformed cells and kill the rest.
- Cloning sites: ideally only one site for each enzyme, so the vector is not cut into many pieces.
- Small size so it enters cells easily.
pBR322 is a common E. coli plasmid with ori, rop, ampR and tetR genes and sites like BamHI and PstI. If we insert a gene inside tetR, that gene stops working (insertional inactivation): recombinant cells grow on ampicillin but die on tetracycline. A simpler test uses the blue-white method: the gene is inserted inside the lacZ gene for β-galactosidase. Colonies with an insert cannot make the enzyme and stay white; those without the insert turn blue on a chromogenic plate.
Vectors for plants: the Ti plasmid of Agrobacterium tumefaciens (disarmed so it no longer causes tumours). For animals: disarmed retroviruses.
4. Competent host
DNA is water-loving and cannot pass the cell membrane easily. We make cells competent: treat bacteria with calcium chloride, chill on ice, heat to 42°C briefly (heat shock), then ice again. Other ways: micro-injection (animal cell nucleus), gene gun / biolistics (plant cells shot with gold or tungsten beads coated with DNA), and disarmed pathogens as carriers.
Steps of recombinant DNA technology
- Isolate the DNA: break the cell with enzymes (lysozyme for bacteria, cellulase for plants, chitinase for fungi). Remove RNA with ribonuclease and proteins with protease. Add chilled ethanol: pure DNA comes out as white threads (spooling).
- Cut the DNA with a restriction enzyme at a fixed temperature. Check the cut with gel electrophoresis.
- Make many copies with PCR (Polymerase Chain Reaction): two short primers + Taq polymerase (heat-stable, from the bacterium Thermus aquaticus). Each cycle = denaturation (~94°C), annealing of primers (~50–60°C), extension (~72°C). After n cycles, 1 DNA → 2ⁿ copies.
- Ligate the gene into the vector (same enzyme on both) with DNA ligase → recombinant DNA.
- Insert into a host (transformation) and select transformed cells with the marker.
- Get the foreign gene product: the protein made by the foreign gene is a recombinant protein. For large amounts, cells grow in bioreactors (100–1000 litre tanks) that keep temperature, pH, oxygen, nutrients and foam under control. The stirred-tank bioreactor has a stirrer and a sparger (to bubble air).
- Downstream processing: separate and purify the product, add preservatives, test (clinical trials for medicines) and pack.
Gel electrophoresis
DNA is negatively charged. In an agarose gel (from seaweed) with an electric field, DNA moves towards the anode (+). The gel acts like a sieve: smaller pieces move farther. Stain with ethidium bromide and see orange bands in UV light. Cut out the band and take the DNA out (elution).
Try it at home: pull out DNA
Mash a ripe banana or some green peas with a pinch of salt and a spoon of dish soap in half a cup of warm water. Filter through a tea strainer. Slowly pour ice-cold rubbing alcohol (ask an adult) down the side of the glass. White threads appear at the top: that is DNA, just like the spooling step. In the 3D, slide the PCR cycles and predict the copies before you look.
Board exam tip: this unit carries about 12 marks. Common questions: features of a vector, pBR322 diagram labels, why same enzyme for vector and gene, steps of PCR, principle of gel electrophoresis, insertional inactivation.
Key formulas and definitions
- Genetic engineering = cut (restriction enzyme) + paste (ligase) + carry (vector) + copy (PCR)
- EcoRI site: 5′-G↓AATTC-3′ (palindrome, sticky ends AATT)
- PCR: copies after n cycles = 2ⁿ × starting copies
- PCR cycle: denature ~94°C → anneal ~55°C → extend ~72°C (Taq polymerase)
- Vector needs: ori + selectable marker + single cloning site + small size
- Gel electrophoresis: DNA (−) moves to anode (+); smaller = farther
- Blue colony = no insert; white colony = recombinant (lacZ inactivated)
Worked examples
1. You start PCR with 1 DNA molecule. How many copies after 5 cycles?
Each cycle doubles. Copies = 2ⁿ = 2⁵ = 32 copies.
2. You start with 10 DNA molecules and run 10 cycles. How many copies?
Copies = 10 × 2¹⁰ = 10 × 1024 = 10,240 copies.
3. How many PCR cycles are needed to get at least 1000 copies from 1 molecule?
2⁹ = 512 (too few), 2¹⁰ = 1024 (enough). So 10 cycles.
4. One PCR cycle takes 3 minutes. How long for 20 cycles, and how many copies from one molecule?
Time = 20 × 3 = 60 min = 1 hour. Copies = 2²⁰ = 1,048,576, about one million.
5. A 6-letter recognition site appears on average once every 4⁶ letters. How often is that? About how many cuts in a DNA of 40,960 base pairs?
4⁶ = 4096. So about one site every 4096 bp. Cuts ≈ 40,960 ÷ 4096 = 10 cuts (on average).
6. A circular plasmid has 3 sites for BamHI. How many pieces after cutting with BamHI? And a straight (linear) DNA with 3 sites?
A circle cut at 3 points gives 3 pieces. A straight line cut at 3 points gives 3 + 1 = 4 pieces. This is why a vector should have only one site for an enzyme.
7. A gene is inserted at the BamHI site inside tetR of pBR322. Which plates do the recombinant cells grow on?
tetR is broken (insertional inactivation), ampR still works. So recombinant cells grow on ampicillin but NOT on tetracycline. Non-recombinant plasmid cells grow on both.
8. Three DNA pieces of 500 bp, 2000 bp and 5000 bp are run on a gel. Which is nearest to the wells?
Bigger pieces move less through the sieve. So 5000 bp stays nearest the wells; 500 bp goes farthest towards the anode.
Common mistakes
- Saying DNA moves to the cathode in gel electrophoresis. DNA is negative, so it moves to the anode (+).
- Using different restriction enzymes for gene and vector and expecting sticky ends to match. Use the same enzyme.
- Thinking a blue colony is recombinant. Blue means lacZ still works, so there is NO insert; white is recombinant.
- Writing that restriction enzymes are exonucleases. They cut inside DNA, so they are endonucleases.