Genetic Technology
Restriction enzymes cut; ligase joins; vectors carry. Three tools give molecular biologists the ability to excise a gene from one organism, insert it into a vector, and introduce it into a host cell — producing recombinant organisms that express a foreign gene. PCR amplifies tiny DNA samples to quantities large enough to analyse; gel electrophoresis separates fragments by size; sequencing reveals the order of bases. Together, these techniques underpin gene therapy, genetically modified organisms, and the genetic screening programmes that are transforming clinical medicine.
Recombinant DNA technology
Recombinant DNA technology is the set of techniques used to isolate, copy, modify, and transfer specific genes or DNA sequences between organisms. The key tools are restriction enzymes (cut), DNA ligase (join), and vectors (carry).
Restriction enzymes
Restriction enzymes (restriction endonucleases) are bacterial enzymes that cut DNA at specific short sequences called recognition sites (typically 4–8 base pairs, palindromic sequences). They are the molecular scissors of genetic technology:
- Each restriction enzyme recognises a specific palindromic base sequence (e.g. EcoRI recognises
GAATTC) - They cut the DNA at or near the recognition site by hydrolyising phosphodiester bonds
- Blunt ends: cut straight across both strands — no overhanging bases; harder to ligate
- Sticky ends (cohesive ends): staggered cuts that leave short single-stranded overhangs; these overhangs are complementary and can base-pair with matching sticky ends from any DNA cut with the same enzyme — enabling joining
- The same restriction enzyme must be used to cut both the donor DNA (gene of interest) and the vector — ensuring compatible sticky ends are produced
DNA ligase — joining fragments
DNA ligase forms phosphodiester bonds between the sugar-phosphate backbones of two adjacent DNA fragments. It seals nicks in the DNA backbone and joins the sticky ends of a gene insert to the opened vector:
- Both the gene of interest and the vector (e.g. a plasmid) are cut with the same restriction enzyme, producing complementary sticky ends
- The gene insert and linearised vector are mixed; the complementary sticky ends base-pair by hydrogen bonding (annealing)
- DNA ligase seals the phosphodiester bonds at each join — forming a continuous, recombinant DNA molecule (recombinant plasmid)
- The sticky end sequences are restored; the recombinant plasmid carries the gene of interest flanked by the original restriction sites
Vectors — carriers of recombinant DNA
A vector is a DNA molecule used to carry a gene of interest into a host cell. The vector must be able to replicate within the host. Common vectors:
Small circular DNA molecules found naturally in bacteria. Modified plasmids used as vectors have: (1) an origin of replication; (2) one or more restriction sites for inserting the gene; (3) one or more antibiotic resistance genes used as selectable markers to identify transformed cells; (4) a reporter gene (e.g. a fluorescent protein or lacZ gene) to identify which colonies carry the insert.
Modified viruses that can infect cells and integrate the gene of interest into the host genome. Used in gene therapy where stable integration into the host chromosome is required. Retroviruses can integrate into the host genome; adenoviruses deliver DNA without integration. The viral DNA carrying the gene is used without the harmful pathogenic parts of the virus.
Transformation — introducing DNA into host cells
Transformation is the uptake of foreign DNA (recombinant plasmid) by a host cell. In bacteria:
- Heat shock: bacteria are treated with cold CaCl₂, then briefly heated to 42°C; this makes the membrane temporarily permeable to DNA
- Electroporation: brief electric pulses create transient pores in the membrane, allowing DNA entry
Not all bacteria take up the plasmid. Transformed cells are identified using antibiotic resistance markers: bacteria are grown on medium containing the antibiotic; only transformed cells (carrying the antibiotic resistance gene on the plasmid) survive.
PCR — polymerase chain reaction
PCR amplifies a specific DNA sequence exponentially from a tiny starting sample — even a single molecule of DNA. It requires a thermostable DNA polymerase (Taq polymerase from Thermus aquaticus), two primers, free deoxyribonucleotides (dNTPs), and a thermal cycler:
- Denaturation (~95°C): high temperature breaks hydrogen bonds between complementary bases; the two strands of the double helix separate
- Annealing (~50–65°C): temperature is lowered; short synthetic primers (oligonucleotides complementary to sequences flanking the target region) bind to the single-stranded template by complementary base pairing
- Extension (~72°C): Taq DNA polymerase (heat-stable; optimal at ~72°C) extends from the primers, synthesising a new complementary strand using free dNTPs
After each cycle, the number of DNA molecules doubles. After 30 cycles: 230 ≈ 109 copies. The target region accumulates exponentially.
Ordinary DNA polymerase (from E. coli) would be denatured and inactivated at the 95°C denaturation step. Thermus aquaticus is a bacterium from hot springs and its polymerase (Taq) is thermostable — it survives the high-temperature denaturation step and remains active throughout all cycles. Without it, new polymerase would have to be added after each cycle (expensive and impractical).
Gel electrophoresis
Gel electrophoresis separates DNA fragments by size. It is used to check the products of restriction digestion or PCR, to produce DNA profiles (fingerprints) for forensic and paternity analysis, and to identify restriction fragment length polymorphisms (RFLPs):
- DNA samples are mixed with a loading dye and loaded into wells cut into an agarose gel
- An electric current is applied; DNA is negatively charged (phosphate groups) and migrates toward the positive electrode
- Smaller fragments migrate faster through the gel matrix; larger fragments are retarded by the mesh
- After running, the gel is stained (e.g. with ethidium bromide or SYBR Green) and visualised under UV light; bands appear where DNA has accumulated
- A DNA ladder (set of fragments of known size) is run in an adjacent lane; the size of unknown fragments is determined by comparison
DNA sequencing
Modern DNA sequencing determines the precise order of bases in a DNA fragment. The Sanger (chain-termination) method is the classic technique required for 9700:
- The DNA fragment to be sequenced is denatured into single strands
- A primer anneals to the template strand; DNA polymerase synthesises new strands using normal dNTPs and a small proportion of dideoxyribonucleotides (ddNTPs)
- ddNTPs lack the 3′-OH group needed to form the next phosphodiester bond — when a ddNTP is incorporated, chain extension terminates
- Each ddNTP type (ddATP, ddCTP, ddGTP, ddTTP) is labelled with a different fluorescent dye; random incorporation produces a population of fragments of every length, each terminated at a known base
- Fragments are separated by capillary electrophoresis by size; a detector reads the fluorescent label at the end of each fragment as it passes; the sequence of colours gives the DNA sequence
When inserting a gene into a plasmid using restriction enzymes, why must the same restriction enzyme be used to cut both the gene and the plasmid?
- A. Different enzymes produce blunt ends that cannot be joined
- B. The same enzyme produces complementary sticky ends on both the gene and plasmid, allowing them to base-pair and be joined by ligase
- C. Different enzymes would cut the gene at too many sites
- D. Only one restriction enzyme can recognise palindromic sequences
PCR is used to amplify a specific DNA region from a crime scene blood sample.
(a) Describe the three stages of one PCR cycle, naming the temperatures used and explaining what happens at each stage. [6]
(b) Explain why Taq DNA polymerase is used rather than the DNA polymerase normally found in human cells. [1]
(a) Three PCR stages [6 marks; 2 each]
- Denaturation (~95°C): high temperature breaks the hydrogen bonds between complementary base pairs; the double-stranded DNA separates into two single strands that act as templates [2]
- Annealing (~50–65°C): temperature is lowered; short synthetic primers (complementary to sequences flanking the target region) bind to the single-stranded templates by complementary base pairing [2]
- Extension (~72°C): Taq DNA polymerase binds at the primer and synthesises a new complementary strand in the 5′ to 3′ direction, using free deoxyribonucleotides (dNTPs); both strands are extended simultaneously [2]
(b) Why Taq polymerase [1 mark]
- Taq polymerase is thermostable — it is not denatured by the high temperature (95°C) of the denaturation step; human DNA polymerase would be permanently denatured and inactivated at this temperature [1]
Gene therapy & genetically modified organisms
Gene therapy — treating genetic disease
Gene therapy involves introducing a functional copy of a gene into the cells of a patient with a genetic disease, to correct the effects of a faulty or absent allele. Two broad approaches:
The functional gene is introduced into body (somatic) cells of the patient — not into gametes or germline cells. Changes affect only the treated individual and are not heritable (not passed to offspring).
Example — cystic fibrosis: functional CFTR gene is delivered into airway epithelial cells; normal chloride ion channels are produced; mucus production is reduced. Treatment must be repeated regularly because somatic cells are replaced and the introduced gene is gradually diluted.
The functional gene is introduced into germline cells (gametes, fertilised eggs, or early embryos). Changes are heritable — all cells of the resulting individual carry the new gene, and it can be passed to subsequent generations.
Germline gene therapy is currently not permitted in humans in most countries due to ethical and safety concerns: permanent heritable changes with unknown long-term consequences; lack of consent from the individual whose germline is modified; possibility of off-target effects passed to future generations.
Gene therapy vectors
| Vector type | Mechanism | Advantages | Disadvantages |
|---|---|---|---|
| Viral vectors (retrovirus, lentivirus, adenovirus) | Virus infects target cells; delivers gene; retrovirus integrates gene into host chromosome | High efficiency of gene delivery; retroviral integration gives long-lasting expression | May trigger immune response; integration can disrupt host genes (insertional mutagenesis) — risk of activating oncogenes |
| Non-viral vectors (liposomes, naked DNA) | Liposomes (phospholipid vesicles) fuse with cell membrane, releasing DNA inside; naked DNA is taken up less efficiently | Less likely to trigger immune response; no risk of insertional mutagenesis | Lower efficiency; less stable; gene expression typically transient |
Genetically modified organisms (GMOs)
A genetically modified organism (GMO) is one that has had a gene from another species (or a modified version of its own genes) introduced into its genome using recombinant DNA technology. The inserted gene is called a transgene; the organism is transgenic.
- Identify and isolate the gene of interest from the donor organism
- Insert the gene into a vector (plasmid or viral vector) using restriction enzymes and ligase
- Introduce the recombinant vector into host cells (bacteria: heat shock; plants: Agrobacterium tumefaciens; animals: microinjection or viral vectors)
- Select transformed cells using selectable markers (antibiotic resistance)
- Grow the transformed organisms; verify gene expression
A gene encoding resistance to a broad-spectrum herbicide (e.g. glyphosate) is inserted into crop plants. The herbicide kills weeds but not the resistant crop. Advantages: reduces need for mechanical weeding; can increase crop yields. Concerns: gene may spread to wild relatives (gene flow) creating herbicide-resistant weeds; farmers are dependent on purchasing the herbicide and resistant seeds from the same company; reduced biodiversity in fields treated with broad-spectrum herbicides.
The gene for Bt toxin (insecticidal crystal protein) from the bacterium Bacillus thuringiensis is inserted into cotton or maize plants. Bt toxin is toxic to the larvae of certain pest insects (e.g. bollworm) but not to mammals or most other organisms. Advantages: dramatically reduces insecticide use; lower production costs; less environmental contamination. Concerns: possible evolution of insect resistance to Bt toxin; potential effects on non-target species; concerns about ecological disruption.
Rice (Oryza sativa) is engineered to produce β-carotene (a precursor of vitamin A) in the grain — which normal white rice does not contain. The grain is yellow/orange, hence "golden rice". Two genes from daffodil and one from a bacterium enable the β-carotene biosynthesis pathway in the endosperm. Purpose: address vitamin A deficiency in populations in low-income countries where rice is a dietary staple; vitamin A deficiency causes preventable blindness. Concerns: regulatory approval delays; whether target populations will adopt it; whether it addresses underlying causes of nutritional deficiency.
Ethical considerations for GMOs
- Biodiversity: widespread monoculture of GM crops may reduce wild and non-GM plant diversity
- Gene flow: transgenes could spread via pollen to wild relatives, potentially creating uncontrollable super-weeds or disrupting natural ecosystems
- Unforeseen effects: long-term consequences of consuming GM foods are uncertain for some stakeholders
- Access and equity: GM technology is largely owned by multinational corporations; small farmers may lose autonomy or face increased costs
- Benefits: increased food security, reduced pesticide use, nutritional enhancement; potentially important in climate adaptation
A patient receives somatic gene therapy for a genetic disorder. Which correctly describes a feature of somatic (not germline) gene therapy?
- A. The introduced gene is present in all cells of the patient's body
- B. The therapeutic effect is permanent and heritable by the patient's children
- C. The introduced gene is not passed to offspring because gametes are not modified
- D. Somatic gene therapy requires integration into the germline to produce a lasting effect
Genetic screening & testing
Genetic screening is the testing of individuals or populations to identify those who carry or will develop a particular genetic condition. It uses techniques from recombinant DNA technology, particularly PCR and DNA probes, to detect specific alleles or mutations.
DNA probes
A DNA probe is a short, single-stranded DNA molecule with a known sequence, labelled with a radioactive isotope or a fluorescent dye. Probes are used to detect complementary sequences in a DNA sample by hybridisation:
- DNA from the patient is extracted, amplified by PCR, and denatured into single strands
- The probe (complementary to the target sequence, e.g. the mutant allele) is added and allowed to hybridise under controlled conditions
- If the target sequence is present, the probe binds (complementary base pairing) to its complement
- Excess unbound probe is washed away
- A radioactive signal (detected by autoradiography) or fluorescent signal indicates that the target allele is present
- Separate probes for the normal and mutant alleles identify whether the patient is homozygous normal, heterozygous (carrier), or homozygous mutant
Types of genetic screening
Individuals who may carry one copy of a recessive allele (carriers) can be identified before they have children. Used for autosomal recessive conditions (cystic fibrosis, sickle-cell disease) and X-linked conditions (haemophilia, Duchenne muscular dystrophy).
Process: blood or saliva DNA sample → PCR amplifies the relevant gene region → DNA probes or sequencing identifies alleles → carrier status reported.
Purpose: allow carriers to make informed reproductive decisions; arrange genetic counselling; inform partners about combined risk.
Testing of fetal DNA to determine whether the fetus has a genetic condition. Two main sample methods:
- Amniocentesis: sample of amniotic fluid withdrawn (~16 weeks); contains fetal cells; slight risk of miscarriage (~0.5–1%)
- Chorionic villus sampling (CVS): sample of placental tissue (~10–12 weeks); can be done earlier; slightly higher miscarriage risk
Fetal DNA is extracted from cells, amplified by PCR, and analysed with DNA probes or karyotyping. Results inform parents about the genetic status of the fetus.
Single cells are biopsied from embryos created by IVF at the 8-cell stage; DNA is amplified by PCR and tested for specific alleles. Only embryos that are unaffected (or at least carriers rather than affected) are implanted. Avoids the need for prenatal testing or decisions about established pregnancies.
Entire populations or specific high-risk groups are screened (e.g. newborn bloodspot screening for PKU, congenital hypothyroidism, etc.). Identifies affected individuals early enough to begin treatment before symptoms develop. Neonatal screening for PKU allows early dietary intervention that prevents intellectual disability.
Ethical considerations in genetic screening
Arguments for screening:
- Early diagnosis allows early treatment — better outcomes (e.g. PKU diet prevents disability)
- Informed reproductive choices for carriers and affected individuals
- Reduces suffering associated with late-diagnosed genetic conditions
- Medical research benefits from genetic data
Arguments against / concerns:
- Psychological impact of learning about a genetic condition (anxiety, stigma)
- Potential for discrimination by insurers or employers if genetic data is not kept confidential
- Prenatal diagnosis may lead to selective termination of affected pregnancies — raises questions about disability and the value of life
- Genetic determinism: oversimplification of complex traits; risk of misinterpretation
- Germline gene editing (future prospect) raises concerns about heritable changes without the consent of future generations
The 9700 exam expects balanced arguments: present both sides clearly and acknowledge that ethical positions depend on values, and that the technology itself is neutral.
Topic 19 Practice — Comprehensive
Mixed practice across recombinant DNA, gene therapy, GMOs, and genetic screening.
Which type of cut by a restriction enzyme produces sticky ends, and why are sticky ends useful?
- A. A straight cut through both strands simultaneously; useful because they are easy to join with ligase
- B. A staggered cut that leaves single-stranded overhangs; useful because the complementary overhangs can base-pair with compatible sticky ends from the insert
- C. A cut at one strand only, leaving the other intact; useful because it reduces the chance of damaging the gene
- D. A straight cut through both strands; useful because they prevent re-ligation of the vector without an insert
DNA samples from two individuals are cut with the same restriction enzyme and run on an agarose gel. Individual 1 shows bands at 800 bp and 200 bp. Individual 2 shows a single band at 1000 bp. What does this suggest?
- A. Individual 2 has no DNA at the 800 bp and 200 bp regions
- B. Individual 2 has no restriction site between the 800 bp and 200 bp positions, so the 1000 bp region is not cut; a restriction fragment length polymorphism (RFLP) exists between the individuals
- C. Individual 1 has less DNA than individual 2
- D. Individual 2's gel ran faster, combining the two bands
Recombinant human insulin is produced by inserting the human insulin gene into bacteria. Outline the steps involved in producing a bacterium that expresses human insulin. [8]
- Obtain the human insulin gene (from mRNA via reverse transcriptase to make cDNA, or by cutting from genomic DNA) [1]
- Cut the insulin gene from donor DNA using a restriction enzyme, producing sticky ends [1]
- Cut the plasmid vector with the same restriction enzyme, producing complementary sticky ends [1]
- Mix the insulin gene and cut plasmid; the complementary sticky ends anneal by base pairing [1]
- DNA ligase seals the phosphodiester bonds, producing a recombinant plasmid containing the insulin gene [1]
- Introduce the recombinant plasmid into bacteria by transformation (e.g. heat shock or electroporation) [1]
- Grow bacteria on selective medium containing antibiotic; only bacteria that have taken up the plasmid (with antibiotic resistance gene) will survive [1]
- Screen surviving colonies to confirm the insulin gene is present and expressed (e.g. using DNA probes or testing for insulin protein production) [1]
- Culture transformed bacteria on a large scale; extract and purify insulin protein [1]
Golden rice has been genetically modified to produce β-carotene in the grain.
(a) Explain why golden rice was developed. [2]
(b) Describe ONE concern about the widespread cultivation of golden rice. [2]
(c) Discuss whether the benefits of golden rice outweigh the concerns, giving a balanced argument. [2]
(a) Why developed [2 marks]
- Vitamin A deficiency causes preventable blindness and immune deficiency, particularly in children in low-income countries where polished white rice is the dietary staple [1]
- β-carotene in golden rice acts as a provitamin A precursor; eating golden rice would supplement dietary vitamin A, reducing deficiency-related health problems [1]
(b) One concern [2 marks]
- Gene flow: the transgene encoding β-carotene biosynthesis could spread via pollen to wild rice relatives, with unknown ecological consequences for natural rice populations and biodiversity [1+1]
- Or: corporate ownership of GM seeds may create economic dependency for small-scale farmers; or uncertainty about long-term effects of consuming the GM crop [1+1]
(c) Balanced argument [2 marks]
- For: the potential to prevent preventable blindness and death in millions of children is a significant humanitarian benefit; the technology has undergone extensive safety testing [1]
- Against: alternative approaches (dietary diversification, vitamin A supplementation) exist and may be more sustainable long-term; gene flow concerns require careful management; the underlying problem is poverty and food insecurity, not just nutritional content of rice [1]
Topic 19 — Common Mistakes
- ✂Saying restriction enzymes cut DNA randomlyRestriction enzymes recognise and cut at specific palindromic recognition sequences — they are highly specific, not random. Each enzyme has its own unique recognition site. "Random cutting" is the exact opposite of their function.
- 📋Confusing ligase with restriction enzyme rolesRestriction enzyme = cuts (breaks phosphodiester bonds); ligase = joins (forms phosphodiester bonds). Many candidates write "ligase cuts the DNA" or "restriction enzyme joins fragments". These are opposite functions. Restriction enzymes act first; ligase acts second to seal the joins.
- 📉Forgetting to specify that the SAME restriction enzyme must cut both the gene and the vectorCompatible sticky ends are only produced if the same enzyme cuts both pieces. Saying "a restriction enzyme cuts the DNA and the plasmid" without specifying that it must be the same enzyme loses a mark. The same enzyme = same recognition site = complementary sticky ends.
- ⚡PCR: saying "DNA polymerase" without specifying TaqIn a PCR question, "DNA polymerase" alone is insufficient. You must state Taq DNA polymerase (or thermostable DNA polymerase) and explain that it withstands the denaturation temperature. "Heat-stable DNA polymerase" is also acceptable. Not specifying thermostability is an incomplete answer.
- ✏Saying DNA moves toward the negative electrode in gel electrophoresisDNA is negatively charged (phosphate groups) and migrates toward the positive electrode in gel electrophoresis. Like charges repel: the negative DNA is pushed away from the negative electrode and toward the positive one. This reversal is a very common mistake.
- 👤Saying somatic gene therapy is heritableSomatic gene therapy changes only body cells; gametes are not affected; the change cannot be inherited. Only germline gene therapy is heritable, and this is currently not permitted in humans. Many candidates confuse the two in answers about cystic fibrosis gene therapy.
- 🌿Saying Bt toxin kills all insectsBt toxin is specific to certain insect larvae (e.g. lepidopteran or coleopteran pests, depending on the specific Bt toxin protein); it is not toxic to humans, most other animals, or most non-target insects (including many beneficial insects at normal exposure levels). Saying it "kills all insects" or "is toxic to animals" is inaccurate.
- 🖥Confusing Sanger sequencing and gel electrophoresisGel electrophoresis separates DNA fragments by size in an agarose gel stained with intercalating dye. Sanger (chain-termination) sequencing uses ddNTPs to terminate chains at specific bases, then separates fragments by capillary electrophoresis and detects fluorescent labels to read the sequence. They both use electrophoresis, but the purpose, method, and readout are different.
- 🔍Saying DNA probes directly repair mutationsDNA probes are detection tools: they identify whether a specific allele or sequence is present in a sample. They do not repair, replace, or change DNA. Probes hybridise to complementary sequences and signal their presence. Gene therapy (vectors carrying functional genes) is what introduces new DNA into cells.
Topic 19 is mechanism-heavy with high essay mark potential. Highest-yield items: restriction enzyme recognition sites + sticky ends (same enzyme for gene and vector), ligase forms phosphodiester bonds, plasmid vector features (origin of replication / antibiotic resistance marker / restriction site), PCR three steps with temperatures and enzymes (denaturation 95°C / annealing 50-65°C / extension 72°C with Taq), gel electrophoresis: negative charge → positive electrode / small fragments move further, Sanger sequencing ddNTP chain termination, somatic vs germline gene therapy (somatic = not heritable / germline = heritable + not permitted in humans), viral vs non-viral vectors advantages/disadvantages, Bt cotton + golden rice examples with benefits and concerns, DNA probe hybridisation mechanism, prenatal screening methods (amniocentesis vs CVS). Synoptic links: Topic 6 (DNA structure / transcription / translation), Topic 16B (PCR amplifies genetic material for analysis), Topic 11 (monoclonal antibodies — another application of cell technology).