Principles of biotechnology and recombinant DNA
Curd, bread and wine are old, microbe-based biotechnology. Making human insulin in bacteria is the modern version: we move a gene from one organism to another.
Biotechnology is the use of living organisms, cells or enzymes to make products and processes on a large scale. Its two core techniques are genetic engineering (altering genetic material) and bioprocess engineering (keeping sterile conditions for growing cells). In 1972 Stanley Cohen and Herbert Boyer made the first recombinant DNA by joining an antibiotic-resistance gene to a plasmid of Salmonella typhimurium.
In the figure : The foreign DNA must be linked to an origin of replication so that it can multiply.
Rule 1: Why an origin of replication is needed — A piece of alien DNA cannot replicate on its own. It is linked to the ori of a vector; then it multiplies in the host (cloning).
- Easy : Alien DNA needs a vector.
- Medium : A plasmid has its own ori.
- Exam-type : Q: Why is alien DNA joined to a vector? A: To get an origin of replication so that it can multiply in the host.
Rule 2: Two techniques — Genetic engineering creates the recombinant DNA and transfers it into the host. Bioprocess engineering maintains sterile (contamination-free) conditions in a bioreactor so that only the desired microbe or cell grows, making large quantities of products like enzymes, vaccines and antibiotics.
- Easy : Sterile conditions are needed in a bioreactor.
- Medium : Without bioprocess engineering only small quantities are made.
- Exam-type : Q: What does bioprocess engineering ensure? A: A contamination-free environment for the growth of the desired cells.
Cohen and Boyer, 1972: the first recombinant DNA, with a gene for antibiotic resistance in a plasmid of Salmonella typhimurium.
Thinking genetic engineering only means cutting DNA. It includes cutting, joining, transferring and expressing genes.
Memory clue: I-I-M — Identify, Introduce, Maintain.
Spot the difference
| Point | Genetic engineering | Bioprocess engineering |
|---|---|---|
| Role | Alters genetic material | Maintains sterile conditions |
| Result | Recombinant organism | Large-scale product |
| Example | Making rDNA | Growing cells in a bioreactor |
Practice questions
- 1Define biotechnology.
- 2Who made the first recombinant DNA?
- 3What does ori stand for?
- 4Name the two core techniques of modern biotechnology.
- 5Which bacterium's plasmid was used by Cohen and Boyer?
- 6What is a recombinant DNA?
- 7Why is a sterile environment essential in biotechnology?
- 8Why must the foreign DNA be introduced along with a vector?
Show answers (try them yourself first)
- 1Use of living organisms, cells or enzymes for large-scale production of useful products.
- 2Stanley Cohen and Herbert Boyer (1972).
- 3Origin of replication.
- 4Genetic engineering and bioprocess engineering.
- 5Salmonella typhimurium.
- 6A DNA formed by joining DNA from two different sources.
- 7Contaminating microbes would compete with and spoil the desired culture.
- 8The vector carries the ori and helps in multiplication and selection in the host.
Restriction enzymes and DNA ligase
Scissors and glue are the most basic tools; in DNA technology, restriction enzymes are the scissors and DNA ligase is the glue.
Restriction endonucleases cut DNA at specific palindromic sequences. The first, Hind II, always cut at a specific six-base-pair sequence. Names follow a rule: the first letter is from the genus and the next two from the species (EcoRI is from Escherichia coli RY13; R is from the strain). Cuts leave overhanging sticky ends, which pair easily; DNA ligase joins them.
In the figure : Fragments cut with the same enzyme have the same sticky ends and join easily.
In the figure : Restriction enzymes are endonucleases.
Rule 1: Palindromic recognition — A palindrome in DNA reads the same on both strands when read in the 5′→3′ direction. EcoRI cuts between G and A at the sequence 5′-GAATTC-3′ on both strands, leaving single-stranded overhangs.
- Easy : GAATTC is the EcoRI site.
- Medium : Cuts are staggered, so sticky ends appear.
- Exam-type : Q: What are sticky ends? A: Single-stranded overhangs on cut DNA that base-pair with complementary ends.
Rule 2: Separating and isolating fragments — Cut DNA fragments are separated by gel electrophoresis in an agarose gel: negatively charged DNA moves to the positive electrode, and smaller pieces move farther. The bands are stained with ethidium bromide and seen under UV light; the band is cut out and purified (elution).
- Easy : DNA is negatively charged.
- Medium : Smaller fragments travel farther in the gel.
- Exam-type : Q: Why is UV light used? A: The ethidium bromide-stained DNA shows bright orange bands under UV light.
Restriction enzyme = molecular scissors; DNA ligase = molecular glue. Gel electrophoresis separates DNA by size.
Thinking DNA moves to the negative electrode. It moves to the positive electrode.
Memory clue: Scissors – Glue — Restriction enzyme cuts; ligase joins.
Spot the difference
| Point | Restriction enzyme | DNA ligase |
|---|---|---|
| Action | Cuts DNA | Joins DNA |
| Site | Specific palindromic sequence | Sticky or blunt ends |
| Called | Molecular scissors | Molecular glue |
Practice questions
- 1What is the first letter of the name EcoRI derived from?
- 2What is a palindromic sequence?
- 3Which enzyme joins DNA fragments?
- 4What kind of ends are made by EcoRI?
- 5Which gel is used for separating DNA fragments?
- 6Which stain is used to see DNA?
- 7Why do two DNA fragments cut with the same enzyme join easily?
- 8Why do smaller DNA fragments travel farther in electrophoresis?
Show answers (try them yourself first)
- 1The genus Escherichia.
- 2A DNA sequence that reads the same on both strands in 5′→3′ direction.
- 3DNA ligase.
- 4Sticky ends.
- 5Agarose gel.
- 6Ethidium bromide.
- 7They have identical sticky ends that base-pair.
- 8They face less resistance in the agarose mesh.
Cloning vectors and competent host
A courier needs an address label and a way to check delivery. A vector carries a gene and carries markers so scientists can tell whether it was delivered.
A vector is a DNA molecule used to carry a gene into a host. Plasmids and bacteriophages are common vectors. A good vector has an origin of replication (ori), a selectable marker (antibiotic-resistance genes), cloning sites (unique recognition sites) and is small. The plasmid pBR322 has ampicillin and tetracycline resistance genes. In insertional inactivation an inserted gene disrupts a marker.
In the figure : In the blue–white screening, colourless colonies carry the recombinant plasmid.
Rule 1: Selection of recombinants — Insertional inactivation: if the foreign DNA is inserted in the gene for β-galactosidase, the enzyme is inactive and the colony stays colourless; non-recombinants give blue colonies. In pBR322, insertion in the tetracycline gene makes cells ampicillin-resistant but tetracycline-sensitive.
- Easy : Blue colony = non-recombinant.
- Medium : Colourless colony = recombinant.
- Exam-type : Q: What is insertional inactivation? A: Loss of function of a marker gene because foreign DNA is inserted in it.
Rule 2: Vectors for plants and animals; getting DNA inside — Agrobacterium tumefaciens with the Ti plasmid, disarmed, is used for plant cells; retroviruses (disarmed) are used for animal cells. Bacterial cells are made competent by treating them with divalent calcium ions, then a heat shock at 42 °C. Other methods: microinjection (animal cells), gene gun or biolistics (plant cells with gold or tungsten particles).
- Easy : Calcium chloride makes bacteria competent.
- Medium : Gene gun shoots DNA-coated gold particles.
- Exam-type : Q: What is the use of the heat shock? A: It makes the bacteria take up the recombinant DNA.
Competent cells take up DNA easily. Ti plasmid: plants; retrovirus: animal cells; gene gun: plant cells; microinjection: animal cells.
Mixing up the colours. Blue = non-recombinant (enzyme active); colourless = recombinant.
Memory clue: Blue → Bare, White → With insert — Blue colonies have no insert; white colonies have the insert.
Spot the difference
| Point | Microinjection | Gene gun |
|---|---|---|
| Used for | Animal cells | Plant cells |
| Method | DNA injected into nucleus | DNA-coated gold particles shot in |
| Other name | — | Biolistics |
Practice questions
- 1What is a vector?
- 2Name two features of a good cloning vector.
- 3Which plasmid has ampicillin and tetracycline resistance genes?
- 4Which vector is used for plant cells?
- 5What temperature is used for heat shock?
- 6What is meant by competent cells?
- 7Why is a selectable marker necessary?
- 8Why are recombinant colonies colourless in the blue–white screening?
Show answers (try them yourself first)
- 1A DNA molecule that carries a foreign gene into a host.
- 2Ori and selectable marker (also cloning sites).
- 3pBR322.
- 4Agrobacterium tumefaciens (Ti plasmid).
- 542 °C.
- 6Cells made ready to take up foreign DNA.
- 7It identifies and removes non-transformed cells.
- 8The inserted gene inactivates the β-galactosidase gene.
Processes of recombinant DNA technology and PCR
A single hair at a crime scene has very little DNA. PCR makes billions of copies so that it can be studied.
The steps are: isolation of DNA (enzymes lysozyme in bacteria, cellulase in plants, chitinase in fungi break the cell; RNase removes RNA; protease removes proteins; chilled ethanol precipitates DNA), cutting with restriction enzymes, amplification by PCR, insertion into a host, and obtaining the product.
In the figure : Repeated cycles of denaturation, annealing and extension amplify the gene of interest many times.
Rule 1: PCR (polymerase chain reaction) — Two sets of primers (short chemically synthesised oligonucleotides) bind to the template; Taq polymerase, from the bacterium Thermus aquaticus, remains active at high temperature and extends the primers using dNTPs. After many cycles about a billion copies are made. The amplified fragment is then used for ligation with a vector.
- Easy : Taq polymerase is heat-stable.
- Medium : Primers are short DNA sequences.
- Exam-type : Q: Why is Taq polymerase used? A: It does not become inactive at the high temperature of denaturation.
Rule 2: Bioreactors and downstream processing — The host cells with the recombinant gene express the protein when they grow. Large cultures (100–1000 litres) are grown in bioreactors (stirred-tank type, sparged with sterile air, with a stirrer, oxygen delivery, foam control, temperature and pH control and sampling ports). Downstream processing means separation and purification of the product before marketing, with clinical trials and quality control.
- Easy : Bioreactor controls temperature and pH.
- Medium : Downstream processing includes purification.
- Exam-type : Q: What is downstream processing? A: Separation and purification of the product before sale.
PCR steps: denaturation → annealing → extension. Taq polymerase is from Thermus aquaticus.
Thinking that primers are enzymes. Primers are short pieces of DNA; Taq polymerase is the enzyme.
Memory clue: D-A-E — Denature, Anneal, Extend.
Spot the difference
| Point | Denaturation | Annealing |
|---|---|---|
| Action | Strands separate | Primers bind |
| Temperature | High (about 94 °C) | Lower |
| Next step | Annealing | Extension |
Practice questions
- 1What does PCR stand for?
- 2Name the enzyme used in PCR.
- 3From which bacterium is Taq polymerase obtained?
- 4What are primers?
- 5Which enzyme breaks plant cells during DNA isolation?
- 6What is used to precipitate DNA?
- 7Why is a bioreactor used?
- 8Why is PCR useful in the early detection of disease?
Show answers (try them yourself first)
- 1Polymerase chain reaction.
- 2Taq polymerase.
- 3Thermus aquaticus.
- 4Short single-stranded DNA pieces that start DNA synthesis.
- 5Cellulase.
- 6Chilled ethanol.
- 7To grow large quantities of cells under controlled conditions to get a lot of product.
- 8It amplifies tiny amounts of pathogen DNA so it can be detected.