Principles and applications of gene technology

Unit 10 Principles and applications of gene technology

Key unit competence: Compare the advantages and disadvantages of gene technology in present-day life.

Key ideas

• Biotechnology is the use of living organisms or their products in industry, agriculture and medicine (brewing, bread, yoghurt, antibiotics). Gene technology (genetic engineering) is the deliberate manipulation of an organism's genes, usually by transferring a gene from one organism to another to produce a genetically modified organism (GMO) or transgenic organism. Recombinant DNA (rDNA) is DNA made by joining DNA from two different sources.
• Steps in gene transfer: (1) obtain the gene — cut it from DNA with restriction enzymes, make it from mRNA using reverse transcriptase, or synthesise it artificially; (2) insert it into a vector (plasmid or virus); (3) transfer the vector into host cells (transformation); (4) identify the cells that took up the gene (markers); (5) clone/grow the transformed cells and express the gene.
• Enzymes: restriction endonucleases cut DNA at specific recognition sequences (often palindromic, e.g. EcoRI cuts G↓AATTC), producing sticky ends (staggered, unpaired bases) or blunt ends; DNA ligase joins sugar–phosphate backbones of fragments; reverse transcriptase (from retroviruses) makes complementary DNA (cDNA) from mRNA — useful because cDNA has no introns, so bacteria can express it; DNA polymerase (e.g. heat-stable Taq polymerase) copies DNA.
• Plasmids are small circular DNA in bacteria: replicate independently, can be cut with one restriction enzyme, carry antibiotic-resistance genes or marker genes (e.g. GFP — fluorescent; lacZ) used to identify transformed bacteria, and are taken up by bacteria treated with calcium ions and heat shock. Artificial DNA synthesis: short DNA sequences can be made from nucleotides by machines (oligonucleotides, primers, probes), using the known amino acid or base sequence.
• PCR (polymerase chain reaction) amplifies DNA in vitro. Mix: target DNA, primers (short single-stranded DNA complementary to the ends of the target), free nucleotides, Taq polymerase, buffer. Cycle: denaturation at about 95 °C (strands separate as H bonds break); annealing at about 55–65 °C (primers bind); extension at about 72 °C (Taq adds nucleotides). Each cycle doubles the DNA: after n cycles, 2ⁿ copies. Uses: forensic samples, diagnosis (HIV, COVID-19), paternity tests, cloning genes, research.
• Other methods of gene transfer: viral vectors (gene therapy), Agrobacterium tumefaciens (Ti plasmid, for plant cells), gene gun (DNA-coated gold particles fired into cells), electroporation (electric pulses open pores), microinjection (into a nucleus of an egg), liposomes (lipid spheres fuse with membranes), heat shock with Ca²⁺.
• Gel electrophoresis separates DNA fragments by size: DNA is cut with restriction enzymes, loaded into wells in agarose gel, and an electric current is applied; DNA is negatively charged (phosphate groups) so it moves towards the positive electrode (anode); smaller fragments move further. Bands are made visible with stains, fluorescent dyes or radioactive probes (Southern blotting). Used in DNA profiling (fingerprinting) for forensics and paternity, based on variable repeated sequences (VNTRs/STRs).
• Microarrays (DNA chips): thousands of known DNA probes fixed on a glass slide; labelled DNA or cDNA from mRNA is added; spots that hybridise fluoresce. Used to compare genomes, detect mutations and find which genes are expressed (active) in a cell, e.g. cancer cells versus normal cells. Bioinformatics uses computers and databases to store and analyse sequences of DNA and proteins (e.g. GenBank, BLAST), compare species and design drugs.
• Applications: medicine — human insulin made by GM bacteria (E. coli) or yeast (no animal sources, no allergic reactions, unlimited supply), growth hormone, clotting factor VIII, vaccines (hepatitis B), gene therapy (inserting normal alleles into cells, e.g. for SCID, cystic fibrosis), genetic screening; agriculture — Bt crops (insect resistant), herbicide-resistant soya, Golden Rice (β-carotene for vitamin A), drought-tolerant and virus-resistant crops (e.g. cassava resistant to brown streak), GM animals producing proteins in milk; forensic science — DNA profiling of crime scene samples; industry — enzymes for detergents, bioremediation.
• Risks and ethics: possible spread of transgenes to wild relatives ("superweeds"), harm to non-target insects, loss of biodiversity, development of pest resistance, unknown health effects (allergies), antibiotic marker genes, dependence of farmers on seed companies (patents, cost), ethical concerns about "playing God", animal welfare, privacy of genetic information, designer babies, unequal access. Regulation: biosafety laws, labelling, risk assessment (Rwanda has biosafety guidelines). Benefits must be weighed against risks.

Common misconceptions

• "Restriction enzymes cut DNA randomly." No: each cuts at a specific recognition sequence.
• "In electrophoresis, larger fragments move further." No: smaller fragments move further through the gel.
• "PCR makes proteins." No: PCR copies DNA; it does not translate.
• "All GM food is dangerous." No: approved GM foods undergo safety testing; risks are assessed case by case.

Worked examples

Practice questions

1. Define gene technology and recombinant DNA.

   Show answer

   Gene technology: manipulation of an organism's genes, e.g. transferring genes between species. Recombinant DNA: DNA formed by combining DNA from different sources.

   Common mistake: Confusing with selective breeding.

2. Describe the roles of restriction enzymes, DNA ligase and reverse transcriptase.

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   Restriction enzymes cut DNA at specific sequences producing sticky ends; ligase joins sugar-phosphate backbones of fragments; reverse transcriptase makes cDNA from mRNA.

   Common mistake: Saying ligase cuts DNA.

3. State four properties of plasmids that make them good vectors.

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   Small and circular; replicate independently; have restriction sites; carry marker genes; taken up by bacteria; copied many times per cell.

   Common mistake: Saying plasmids are in human cells.

4. Describe the stages of PCR.

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   Denaturation at 95 °C separates strands; annealing at 55–65 °C primers bind; extension at 72 °C Taq polymerase adds nucleotides; cycle repeated, doubling DNA each time.

   Common mistake: Mixing up temperatures.

5. Explain how gel electrophoresis separates DNA fragments.

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   DNA cut and placed in wells; current applied; negatively charged DNA moves to anode; agarose acts as sieve; smaller fragments move further; stained bands visualised.

   Common mistake: Saying DNA is positively charged.

6. Describe how human insulin is produced by gene technology.

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   mRNA from β cells; reverse transcriptase makes cDNA; sticky ends added; plasmid cut with same restriction enzyme; ligase joins; plasmid into E. coli; select with markers; culture in fermenters; extract and purify insulin.

   Common mistake: Using genomic DNA with introns.

7. State four applications of gene technology.

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   Insulin and other proteins; vaccines; gene therapy; GM crops (Bt, Golden Rice); DNA profiling; diagnosis of genetic disease; bioremediation.

   Common mistake: Listing only one field.

8. Discuss three advantages and three concerns of GM crops.

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   Advantages: higher yields, pest and drought resistance, less pesticide, better nutrition. Concerns: gene flow to wild plants, harm to non-target species, pest resistance, allergies, seed costs and patents, ethics.

   Common mistake: Giving only one side.

Matching and drag-and-drop

1. Matching

   Match each item in column A with the correct answer in column B. Write the letter next to each number.

   Column A	Column B
   1. A small circular DNA molecule in bacteria is a:	a. plasmid
   2. Using electric pulses to open pores in membranes is:	b. gene gun method
   3. Firing DNA-coated gold particles into cells is the:	c. electroporation
   4. Short DNA strands that start DNA copying in PCR are:	d. primers

   Show answer

   1 → a (plasmid) · 2 → c (electroporation) · 3 → b (gene gun method) · 4 → d (primers)

   • Used as a vector.
   • DNA enters.
   • Biolistics.
   • Bind target ends.

2. Drag and drop

   Choose the correct word or phrase from the box for each gap. One word in the box is not needed.

   calcium ions and heat shock  ·  1 024 copies  ·  reverse transcriptase  ·  100 copies
   1. Bacteria take up plasmids more easily after .
   2. After 10 PCR cycles one DNA molecule becomes .
   3. cDNA is made from mRNA by .
   Show answer

   1: calcium ions and heat shock · 2: 1 024 copies · 3: reverse transcriptase

   • Transformation.
   • 2¹⁰.
   • From retroviruses.

3. Matching

   Match each item in column A with the correct answer in column B. Write the letter next to each number.

   Column A	Column B
   1. In PCR, DNA strands separate at about:	a. 95 °C
   2. Golden Rice was modified to produce:	b. DNA ligase
   3. The enzyme that joins DNA fragments is:	c. β-carotene
   4. An organism with a gene from another species is:	d. transgenic

   Show answer

   1 → a (95 °C) · 2 → c (β-carotene) · 3 → b (DNA ligase) · 4 → d (transgenic)

   • Denaturation.
   • Vitamin A precursor.
   • Seals the backbone.
   • A GMO.

4. Drag and drop

   Choose the correct word or phrase from the box for each gap. One word in the box is not needed.

   positive electrode  ·  most charged per molecule  ·  smallest  ·  72 °C
   1. In gel electrophoresis DNA moves towards the .
   2. Extension in PCR occurs at about .
   3. In electrophoresis the fragments that travel furthest are the .
   Show answer

   1: positive electrode · 2: 72 °C · 3: smallest

   • DNA is negative.
   • Optimum for Taq.
   • Pass gel pores easily.

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