explain the roles of restriction endonucleases, DNA ligase, plasmids, DNA polymerase and reverse transcriptase in the transfer of a gene into an organism

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

Learning Objective

Explain the roles of restriction endonucleases, DNA ligase, plasmids, DNA polymerase and reverse transcriptase in the transfer of a gene into an organism, and describe the associated vectors, host cells, methods, screening techniques, applications and ethical considerations. (AO1 – knowledge; AO2 – understanding; AO3 – application)

1. Molecular Tools Used in Gene Transfer

1.1 Restriction Endonucleases (AO1, AO2)

• Proteins that recognise short, specific DNA sequences (4–8 bp) and cleave the phosphodiester backbone.
• Types of cuts:

  • Sticky (cohesive) ends – over‑hanging single‑stranded DNA that can base‑pair with a complementary sequence.
  • Blunt ends – straight cuts with no over‑hangs.

• In recombinant DNA work they are used to cut both the vector and the gene of interest, creating compatible ends for ligation.

1.2 DNA Ligase (AO1, AO2)

• Catalyses the formation of phosphodiester bonds between the 5′‑phosphate of one DNA fragment and the 3′‑hydroxyl of another.
• Joins both sticky‑ended and blunt‑ended fragments (sticky ends ligate more efficiently).
• Produces a stable recombinant DNA molecule ready for introduction into a host.

1.3 DNA Polymerases (AO1, AO2)

• Thermostable Taq polymerase – high activity, low fidelity; ideal for routine PCR when sequence accuracy is not critical.
• High‑fidelity polymerases (e.g., Pfu, Phusion) – possess proofreading activity; essential for cloning and downstream expression.
• Roles in gene transfer:

  • Polymerase‑Chain Reaction (PCR) – amplifies the target gene to obtain sufficient quantity for cloning.
  • Repair of nicks – fills in missing nucleotides at sticky‑end junctions before ligase seals the backbone.

1.4 Reverse Transcriptase (AO1, AO2)

• Synthesises complementary DNA (cDNA) from an RNA template.
• Important for:

  • Converting eukaryotic mRNA (which may contain introns) into intron‑free cDNA suitable for expression in prokaryotic hosts.
  • Generating cDNA libraries for screening and cloning of specific genes.

2. Vectors for Gene Transfer (AO1, AO2)

3. Host Cells & Introduction Methods (AO1, AO2, AO3)

• Bacterial hosts (E. coli)

  • Transformation by CaCl₂‑competent cells – heat‑shock (42 °C, 45 s) gives typical efficiencies of 10⁶ – 10⁸ cfu µg⁻¹ DNA.
  • Electroporation – brief high‑voltage pulse; efficiencies up to 10⁹ cfu µg⁻¹.

• Yeast (S. cerevisiae)

  • Lithium acetate / PEG method – yields 10³ – 10⁴ transformants µg⁻¹.
  • Electroporation – higher efficiencies for large plasmids.

• Plant cells

  • Agrobacterium‑mediated transformation – T‑DNA transfer; widely used for dicots.
  • Biolistic (gene‑gun) delivery – DNA‑coated gold/tungsten particles; suitable for monocots and cereal crops.

• Mammalian cells

  • Calcium‑phosphate precipitation.
  • Liposome‑mediated delivery (lipofection).
  • Electroporation.
  • Viral vectors (see above).

4. Polymerase‑Chain Reaction (PCR) – Theory & Practice (AO1, AO2, AO3)

PCR is the cornerstone technique for obtaining large quantities of a specific DNA fragment.

4.1 Essential Components

• Template DNA.
• Two short primers (forward & reverse) – 18–25 nt, 40–60 % GC, T_m 50–65 °C, no significant secondary structures.
• DNA polymerase (Taq for speed; high‑fidelity for cloning).
• dNTPs, MgCl₂, buffer, and optional additives (e.g., DMSO for GC‑rich templates).

4.2 Cycle Stages

1. Denaturation – 94–98 °C; separates strands.
2. Annealing – 50–65 °C; primers bind to complementary sites.
3. Extension – 72 °C; polymerase adds nucleotides (≈1 kb min⁻¹).

4.3 Practical Tips (AO2)

• Mg²⁺ concentration influences enzyme activity and fidelity – usually 1.5–2.5 mM.
• Number of cycles: 25–35 (each cycle doubles the product).
• Use a “hot‑start” polymerase to reduce non‑specific amplification.
• For cloning, include restriction sites in the 5′ ends of primers.

4.4 Quantitative PCR (qPCR) (AO2)

Real‑time monitoring using SYBR Green or TaqMan probes; provides quantitative data on gene expression.

4.5 Key Equation (placed with other equations)

Amount of DNA after n cycles:

\$N = N_0 \times 2^{n}\$

5. Screening & Confirmation of Recombinant Clones (AO1, AO2, AO3)

• Blue‑white screening – lacZ α‑complementation; insertion of a gene disrupts β‑galactosidase, producing white colonies on X‑gal/IPTG plates.
• Colony PCR – rapid test using primers flanking the MCS.
• Restriction analysis – isolate plasmid, digest with diagnostic enzymes, compare band pattern on agarose gel.
• DNA sequencing – definitive confirmation of insert identity and orientation.
• Southern blotting – used for large constructs (BACs, YACs) to verify integration and copy number.
• Phenotypic screening – antibiotic resistance, fluorescence (GFP), enzymatic colour change.

6. Step‑by‑Step Workflow for a Typical Gene‑Transfer Experiment (AO1‑AO3)

1. Gene isolation – PCR amplification of the target gene or synthesis of cDNA using reverse transcriptase.
2. Vector selection – choose a plasmid or other vector with appropriate promoter, selectable marker and MCS.
3. Restriction digestion – cut both insert and vector with compatible enzymes (e.g., EcoRI + HindIII).
4. Purification – gel‑extraction or column purification of the digested fragments.
5. Ligation – mix insert and vector; allow sticky ends to anneal; add DNA ligase (typically 1 h at 16 °C).
6. Transformation / Transfection

   • Bacterial transformation – heat‑shock or electroporation.
   • Yeast / plant transformation – lithium acetate or Agrobacterium.
   • Mammalian transfection – lipofection, calcium‑phosphate, or viral infection.

7. Selection – plate on medium containing the appropriate antibiotic or screen for reporter activity.
8. Screening – blue‑white screening, colony PCR, restriction analysis, or sequencing.
9. Confirmation of expression – SDS‑PAGE, Western blot, enzyme assay, fluorescence microscopy, etc.

7. Real‑World Applications (AO1, AO2)

• Recombinant insulin – human insulin gene cloned into a pBR322‑derived plasmid and expressed in E. coli.
• Growth hormone (GH) – produced in bacterial or yeast expression systems for therapeutic use.
• GM crops – Bt toxin gene in cotton/maize; glyphosate‑resistant wheat (EPSPS gene).
• Gene therapy – AAV vectors delivering functional RPE65 for Leber congenital amaurosis.
• Vaccines – recombinant hepatitis B surface antigen in yeast; mRNA vaccines (cDNA template generated by reverse transcription).
• Genome editing – CRISPR/Cas9, TALENs and ZFNs used for knock‑in/out in plants, animals and human cells.

8. Ethical, Social and Environmental Issues (AO1)

• Labelling of GM foods and consumer choice.
• Potential impact of gene‑driven pest control on non‑target species.
• Intellectual‑property rights versus access to life‑saving medicines.
• Safety of gene‑therapy trials (off‑target effects, germ‑line modifications).
• Bio‑security concerns surrounding the creation of novel pathogens.
• Equity of benefit distribution between developed and developing nations.

9. Emerging Technologies Influencing Genetic Technology (AO1, AO2)

• CRISPR/Cas9 – RNA‑guided nuclease for precise knock‑in, knock‑out and base editing.
• TALENs & Zinc‑Finger Nucleases (ZFNs) – protein‑based DNA‑binding nucleases.
• Synthetic biology – design of whole metabolic pathways (e.g., engineered microbes producing artemisinin).
• RNA‑i and antisense technologies – post‑transcriptional gene silencing without altering DNA.
• Prime editing – installs targeted insertions, deletions and all 12 possible base‑pair conversions without double‑strand breaks.

10. Summary Table – Molecular Tools and Their Primary Functions (AO1, AO2)

11. Key Equations (AO2)

Amount of DNA after n PCR cycles:

\$N = N_0 \times 2^{n}\$

Ligation efficiency (probability of successful ligation of two sticky‑ended fragments):

\$P = 1 - e^{-k\,[\text{DNA}]\,t}\$

• k = rate constant, [\text{DNA}] = concentration of DNA ends, t = incubation time.

12. Suggested Diagram (AO1)