Recombinant DNA technology
Recombinant DNA joins a useful gene into a vector and puts it in a host cell, which then makes the gene's product, such as human insulin.
Steps in making recombinant DNA
Restriction enzymes are the scissors, ligase is the glue, the plasmid is the carrier and the host cell is the factory. Starred steps carry the key enzymes and the selection.
- 1Isolate the gene of interest and the vector DNAhuman genes are often copied from mRNA as cDNA by reverse transcriptase
- 2★Cut the gene and the vector with the same restriction enzymeRestriction endonuclease (e.g., EcoRI) · leaves matching 'sticky ends'
- 3★Join the gene into the vector → recombinant DNADNA ligase · seals the sugar–phosphate backbone
- 4Put the recombinant DNA into host cells (transformation)E. coli or yeast; by heat shock or electroporation
- 5★Select the cells that carry the recombinant DNAmarker genes, e.g., antibiotic resistance
- 6Grow (clone) the selected cells and collect the productlarge fermenters; product is purified
★ one-way, controls the pace
Tools of the trade
| Tool | What it does | Example |
|---|---|---|
| Restriction endonuclease | Cuts DNA at a specific short base sequence, often a palindrome | EcoRI (cuts at GAATTC), HindIII |
| Vector | Carries the gene into the host and copies itself there | Plasmids (small circular DNA in bacteria, e.g., pBR322), bacteriophages, cosmids |
| DNA ligase | Joins DNA pieces by forming phosphodiester bonds | T4 DNA ligase |
| Host cell | Takes up the recombinant DNA, copies it and makes the protein | E. coli (bacterium), Saccharomyces cerevisiae (yeast) |
| Marker gene | Shows which cells carry the vector | Ampicillin and tetracycline resistance genes in pBR322 |
| Reverse transcriptase | Makes DNA (cDNA) from mRNA, giving a gene without introns that bacteria can use | From retroviruses |
Applications in nutrition and health
| Product | What it does |
|---|---|
| Human insulin | Made in E. coli or yeast; replaced insulin from animal pancreas. The first recombinant DNA drug, approved in 1982 |
| Golden rice | Rice given genes (from daffodil or maize and a soil bacterium) so the grain makes β-carotene (provitamin A), to fight vitamin A deficiency |
| Bt crops | Carry a gene from Bacillus thuringiensis that makes an insect-killing (Cry) protein, so less pesticide is sprayed. Bt cotton has been grown in India since 2002 |
| Recombinant vaccines | Hepatitis B vaccine is the viral surface protein (HBsAg) made in yeast, so no whole virus is used |
| Other proteins | Growth hormone, interferons, clotting factor VIII, erythropoietin; recombinant chymosin (rennet) for cheese-making |
Safety and ethics
A moratorium was placed on Bt brinjal in India in 2010, showing how safety debates shape what reaches the market.
Golden rice was made to supply β-carotene (provitamin A) where rice is the staple and vitamin A deficiency is common. Recombinant human insulin is a daily need for many people with diabetes. Not every nutrient-rich crop is GM: iron-rich bajra was made by conventional breeding (biofortification).
2 marks: “What are restriction endonucleases?” Bacterial enzymes that cut DNA at specific base sequences; the 'molecular scissors' of genetic engineering (e.g., EcoRI).
5 marks: “List the applications of recombinant DNA technology in nutrition and health.” Human insulin, golden rice, Bt crops, hepatitis B vaccine, growth hormone and recombinant chymosin, one line each.
10 marks: “Explain the steps in recombinant DNA technology with its applications.” Draw the plasmid cloning diagram, give the six steps with enzymes, then applications and a note on safety.
Scissors, glue, carrier, factory: restriction enzyme, ligase, plasmid, host cell.