explain that genes to be transferred into an organism may be: extracted from the DNA of a donor organism, synthesised from the mRNA of a donor organism, synthesised chemically from nucleotides

Principles of Genetic Technology (Cambridge AS & A Level – Topic 19)

Learning Objective

Explain how a gene that is to be transferred into a recipient organism can be obtained, and describe the subsequent steps required to create a recombinant DNA construct.

1. Sources of a Recombinant Gene

1.1 Extraction from Donor Genomic DNA

• Key idea: The whole genome of the donor is isolated and the gene of interest is cut out with restriction endonucleases.
• Typical steps

  1. Cell lysis – detergent, lysozyme or mechanical disruption releases DNA.
  2. Purification of genomic DNA – phenol‑chloroform extraction or commercial kits.
  3. Restriction digestion – two enzymes that flank the gene generate compatible sticky or blunt ends.
  4. Agarose‑gel electrophoresis – visualise the fragment, excise the band, and purify with a gel‑extraction kit.
  5. Ready for cloning – keep the fragment on ice for ligation into a vector.

• When to use – bacterial or viral genes that contain no introns, or when native regulatory sequences are required.

1.2 Synthesis from Donor mRNA (cDNA)

• Key idea: In eukaryotes the mature mRNA has already had introns removed; reverse‑transcription therefore yields an intron‑free DNA copy.
• Typical steps

  1. RNA extraction – isolate total RNA from tissue where the gene is expressed (e.g., pancreas for insulin).
  2. Reverse transcription – reverse transcriptase synthesises single‑stranded cDNA from the mRNA template.
  3. Second‑strand synthesis – DNA polymerase I (or a commercial kit) converts ss‑cDNA into ds‑cDNA.
  4. PCR amplification – gene‑specific primers (often with added restriction sites) generate many copies.
  5. Cloning – the amplified cDNA is ligated into a vector (see Section 2).

• When to use – eukaryotic genes that contain introns, or when only the coding region is required.

1.3 Chemical Synthesis of Genes

• Key idea: An oligonucleotide synthesiser builds a DNA strand one nucleotide at a time on a solid support.
• Typical steps

  1. Design – write the exact sequence; optimise codons for the intended host and add any tags or restriction sites.
  2. Oligonucleotide synthesis – automated synthesiser produces short overlapping oligos (≈50–100 bp).
  3. Assembly – overlap‑extension PCR or ligation joins the oligos into the full‑length gene.
  4. Verification – Sanger sequencing confirms the assembled sequence before downstream work.

• When to use – gene is unavailable in nature, large‑scale codon optimisation is needed, or precise mutations/tags are required.

1.4 Decision‑Tree: Which Source Is Most Suitable?

2. Vectors – DNA “Vehicles” for the Gene

2.1 Plasmid Vectors (bacterial)

A circular DNA molecule that can replicate independently in a host cell.

Suggested schematic (inserted as a figure in class notes): a circular plasmid drawn clockwise showing ori → MCS → promoter → gene of interest → terminator → antibiotic marker → reporter (optional).

2.2 Viral Vectors

Modified viruses that deliver DNA into eukaryotic cells. They are engineered to be replication‑deficient and to carry a therapeutic or experimental gene.

• Adenovirus – high transduction efficiency, non‑integrating, used for vaccines and gene‑therapy trials.
• Lentivirus (derived from HIV‑1) – integrates into the host genome, useful for stable expression in dividing and non‑dividing cells.
• Adeno‑associated virus (AAV) – low immunogenicity, predominantly episomal, popular for in‑vivo gene therapy.

Safety modifications typically include deletion of essential viral genes and the addition of a “self‑inactivating” (SIN) LTR in lentiviral vectors. Applications include gene‑therapy for cystic fibrosis, cancer immunotherapy, and production of recombinant proteins in mammalian cells.

3. Cloning Strategies – Inserting the Gene into a Vector

3.1 Traditional Restriction‑Enzyme Cloning

1. Digest vector and insert with the same pair of restriction enzymes.
2. De‑phosphorylate the vector (optional) to reduce self‑ligation.
3. Ligate with T4 DNA ligase (typically 1 h at 16 °C or 15 min at room temperature).

3.2 Alternative (Advanced) Cloning Methods

• Gibson Assembly – exonuclease creates overlapping ends, polymerase fills gaps, and ligase seals nicks; useful for joining multiple fragments in a single reaction.
• Golden‑Gate (type IIs) Assembly – uses type IIs enzymes that cut outside their recognition site, allowing seamless, directional assembly of several parts.
• TOPO (TA) Cloning – exploits the 3′‑A overhangs added by Taq polymerase and a topoisomerase‑linked vector for rapid, ligase‑free insertion.

These methods are often featured in “advanced” A‑Level questions that ask for alternatives to restriction‑enzyme cloning.

4. Transformation & Selection of Recombinant Host Cells

4.1 Preparing Competent Cells

• Calcium‑chloride (CaCl₂) method – cells are chilled in 0.1 M CaCl₂, creating pores that allow DNA entry during heat‑shock.
• Rubidium‑chloride (RbCl) method – similar to CaCl₂ but gives slightly higher efficiency for some strains.
• Electro‑competent cells – washed in low‑ionic‑strength buffer (e.g., 10% glycerol) and stored at –80 °C for electroporation.

4.2 Introducing the DNA

• Heat‑shock transformation – 42 °C for 45 s (CaCl₂‑competent cells) followed by recovery in SOC medium.
• Electroporation – a brief 2.5 kV pulse (5 ms) in a cuvette; higher efficiency, especially for large plasmids.

4.3 Selecting Transformants

• Antibiotic markers

  • Ampicillin (AMP) – cheap, but prone to “satellite colonies”.
  • Kanamycin (KAN) – more stable, useful when AMP resistance is already present in the host.
  • Chloramphenicol (CAM) – often used with vectors that have a high‑copy ori.

• Blue‑white screening – vectors with the lacZ α‑fragment allow insertional inactivation; X‑gal/IPTG plates give blue colonies (no insert) and white colonies (insert present).

5. Verification of Recombinant DNA

5.1 Restriction‑Digest Analysis

Isolate plasmid DNA from a few colonies, digest with diagnostic enzymes, and run on an agarose gel. Example:

• Plasmid size = 5 kb; insert = 1.2 kb.
• Digest with Enzyme A (cuts once in the vector) and Enzyme B (cuts at the ends of the insert).
• Expected bands: 5 kb (vector backbone) + 1.2 kb (insert) – total 6.2 kb on the gel.

5.2 Colony PCR

Pick a single colony, add a tiny amount of cells to PCR mix with primers flanking the MCS. A product of the correct size (e.g., 1.2 kb) indicates a successful clone without the need for plasmid purification.

5.3 Sanger Sequencing

• Send purified plasmid with forward and reverse sequencing primers (usually annealing just outside the MCS).
• Interpret chromatograms: clear, non‑overlapping peaks; any mixed peaks suggest heterozygous clones or secondary structures.
• Confirm the entire coding region and any added tags or restriction sites.

6. Real‑World Applications

• Recombinant human insulin – cDNA cloned into a plasmid, expressed in E. coli, purified for diabetes treatment.
• Bt cotton – gene from Bacillus thuringiensis (often PCR‑amplified) inserted into a plant‑transformation vector to confer insect resistance.
• Golden Rice – chemically synthesised phytoene synthase and lycopene β‑cyclase genes stacked to produce β‑carotene in rice endosperm.
• Recombinant human growth hormone (hGH) – cDNA expressed in E. coli or Komagataella phaffii (formerly Pichia pastoris).
• GM salmon (AquAdvantage) – growth‑hormone gene from Chinook salmon driven by an ocean‑type promoter, inserted via plasmid‑mediated transgenesis.
• Gene‑therapy vectors – AAV delivering a functional copy of the RPE65 gene for Leber congenital amaurosis; lentiviral CAR‑T cell therapies for cancer.

7. Ethical, Biosafety & Societal Issues

• Biosafety – containment levels (BSL‑1 to BSL‑3), gene‑flow to wild relatives, development of resistant pests, and horizontal transfer of antibiotic‑resistance markers.
• Labelling & consumer choice – debate over mandatory labeling of GM foods and the impact on market acceptance.
• Intellectual property – patents on specific genes, vectors, or cloning methods can restrict access for research and for low‑income countries.
• Animal welfare – use of transgenic animals for pharmaceutical production (e.g., “pharming” goats producing antithrombin) raises concerns about husbandry and humane treatment.
• Human gene therapy – ethical questions about germ‑line editing, consent, equitable access, and long‑term monitoring of viral vector safety.

8. Comparison of Gene‑Acquisition Methods

9. Skills Checklist – Planning a Cloning Experiment (AO3)

1. Define the aim and select the most appropriate gene‑acquisition method (see decision‑tree).
2. Choose a vector with the required elements (ori, MCS, promoter, selectable marker, optional reporter).
3. Design primers:

   • Include restriction sites or overlapping sequences for the chosen cloning method.
   • Check for hairpins, dimers, and melting‑temperature compatibility.

4. Prepare competent host cells (CaCl₂ or electroporation) and decide on the transformation method.
5. Plan selection:

   • Antibiotic (ampicillin, kanamycin, etc.).
   • Blue‑white screening if using lacZ‑α vectors.

6. Include controls:

   • Negative control – vector only (checks background colonies).
   • Positive control – plasmid known to transform efficiently (confirms competence).

7. Verification steps – restriction digest, colony PCR, and Sanger sequencing with appropriate primers.
8. Record expected results (e.g., band sizes on a gel, PCR product length).
9. Address biosafety: work at the appropriate containment level, dispose of antibiotic‑resistant waste, and obtain any necessary ethical approvals.

10. Key Points to Remember

• All three acquisition routes ultimately give a DNA fragment that can be ligated into a vector.
• Choice of source depends on gene size, presence of introns, availability of donor tissue, need for sequence modification, and cost.
• Core cloning tools: restriction enzymes, ligase, and a suitable vector; advanced tools include Gibson, Golden‑Gate, and TOPO cloning.
• Transformation, selection, and verification are essential to confirm that the recombinant construct is correct and functional.
• Real‑world examples illustrate the impact of genetic technology, while ethical and biosafety considerations must always be part of the experimental design.