define the term recombinant DNA

Principles of Genetic Technology – Recombinant DNA (rDNA)

1. Definition of Recombinant DNA

Recombinant DNA (rDNA) is a molecule of DNA that has been artificially assembled by joining genetic material from two or more different sources. The resulting construct contains a combination of genes that would not naturally occur together in a single organism, enabling the expression of new traits or the production of valuable proteins.

2. Vectors – Vehicles for DNA Transfer

Vectors are DNA molecules that can carry a gene of interest into a host cell, replicate there, and often drive its expression. The choice of vector depends on the size of the DNA fragment, the host organism, and the intended application.

3. Host‑Cell Considerations

Choosing the appropriate host balances growth characteristics, post‑translational modification (PTM) capacity, biosafety, and cost.

4. Elements Required for Transcription and Translation (AO2)

Each element must be justified when designing a recombinant construct – a key requirement of Assessment Objective 2.

• Promoter – initiates transcription. Example: T7 (bacterial), GAL1 (yeast), CMV (mammalian). Why needed? Provides RNA polymerase binding site.
• Ribosome‑binding site (RBS) / Kozak sequence – positions the ribosome at the start codon. Example: Shine‑Dalgarno (bacteria), Kozak consensus (eukaryotes).
• 5′‑UTR & leader sequences – can enhance translation efficiency (e.g., leader peptide in pET vectors).
• Start codon (ATG) and open reading frame (ORF) – defines the protein‑coding region.
• Terminator / poly‑A signal – stops transcription and stabilises mRNA. Example: T7 terminator (bacteria), SV40 poly‑A (mammalian).
• Selectable marker – enables identification of transformed cells (e.g., ampR, kanR, hygromycin B resistance). Why needed? Allows growth on selective medium.
• Reporter gene (optional) – visual confirmation of expression (e.g., GFP, β‑galactosidase).
• Multiple‑cloning site (MCS) – provides several unique restriction sites for easy insertion of the gene of interest.

5. Expression Systems

Table summarises the most frequently used systems at AS & A‑Level, together with typical vectors, promoters, selection markers and special features.

6. Typical Workflow for Creating Recombinant DNA (with QC checkpoints)

1. Isolation of DNA
   • Extract genomic DNA or synthesize cDNA of the target gene.
   • Quantify using a spectrophotometer (A260/A280 ≈ 1.8–2.0) – QC1.
2. Design of Primers / Choice of Restriction Sites
   • Select enzymes that do not cut within the gene.
   • Include 4–6 bp overhangs in primers for sticky‑end cloning.
   • Calculate melting temperatures (Tm) and check for secondary structures – QC2.
3. Restriction Digestion
   • Digest vector and insert with compatible enzymes (e.g., EcoRI + HindIII).
   • Incubate with excess enzyme (1 U/µg DNA) for 1 h at 37 °C.
   • Run a small aliquot on agarose gel to confirm complete digestion – QC3.
4. Purification
   • Gel‑extract the correct bands (use a low‑melting‑point agarose if possible).
   • Elute in TE buffer; measure concentration again.
5. Ligation
   • Set up reactions with insert : vector molar ratios of 3 : 1 (sticky ends) or 1 : 1 (blunt).
   • Include a vector‑only control (no insert) and a no‑ligase control.
   • Incubate at 16 °C overnight or 30 min at room temperature with T4 DNA ligase.
6. Transformation / Transfection
   • E. coli: heat‑shock (42 °C, 45 s) or electroporation.
   • Yeast: lithium acetate/PEG method.
   • Mammalian cells: liposome‑mediated (Lipofectamine) or viral infection.
7. Selection & Screening
   • Plate on medium containing the appropriate antibiotic or auxotrophic supplement.
   • Screen colonies by:
     • Blue/white screening (if lacZ α‑fragment present).
     • Colony PCR using vector‑specific and insert‑specific primers.
     • Restriction‑mapping of miniprep plasmid.
   • Confirm the correct orientation and sequence by Sanger sequencing – QC4.
8. Expression & Analysis
   • Induce expression (e.g., add IPTG 0.5 mM for T7 system) and grow under optimal temperature (often 18–30 °C for soluble protein).
   • Harvest cells, lyse, and analyse protein by SDS‑PAGE.
   • Confirm identity with Western blot or activity assay.
   • Quantify yield (mg protein per L culture) and assess functionality.

7. Applications of Recombinant DNA Technology

7.1 Industrial & Pharmaceutical

• Human insulin, growth hormone, and clotting factors (e.g., Factor VIII) produced in E. coli or CHO cells.
• Enzymes for food processing (amylase, protease) and detergents.
• Monoclonal antibodies (e.g., trastuzumab) and therapeutic enzymes produced in mammalian expression systems.
• Current high‑impact examples: mRNA‑based COVID‑19 vaccines (Pfizer/BioNTech, Moderna) – plasmids encoding the spike protein are transcribed in‑vitro; CRISPR‑Cas9 plasmids used for gene‑editing services.

7.2 Agricultural

• Bt crops – expression of Bacillus thuringiensis Cry toxin for insect resistance.
• Herbicide‑resistant cereals – genes conferring tolerance to glyphosate (EPSPS) or glufosinate (bar).
• Golden Rice – β‑carotene biosynthesis pathway introduced to combat vitamin A deficiency.
• New developments: RNAi‑based pest control (e.g., Bt‑free corn expressing dsRNA) and gene‑edited (CRISPR) wheat with reduced gluten.

7.3 Medical & Therapeutic

• Gene‑therapy vectors (AAV, lentivirus) delivering functional copies of defective genes (e.g., RPE65 for Leber congenital amaurosis).
• Recombinant viral vaccines – adenovirus‑based COVID‑19 vaccine (Oxford/AstraZeneca) and recombinant VSV‑based Ebola vaccine.
• CAR‑T cell therapy – patient T‑cells transduced with a recombinant lentiviral vector encoding a chimeric antigen receptor.
• CRISPR‑Cas9 plasmids for targeted genome editing in research and emerging clinical trials.

8. Ethical, Legal & Social Issues (ELSI)

• Biosafety levels – Level 2 for most plasmid work; Level 3 for certain viral vectors (e.g., replication‑competent adenovirus).
• Regulation of GMOs – EU Directive 2001/18/EC, US USDA/APHIS, and WHO guidelines for clinical gene‑therapy trials.
• Intellectual‑property rights – Patents on genes, vectors, and specific engineered traits; debate over “gene ownership”.
• Public perception & labeling – Mandatory labeling in the EU; voluntary labeling elsewhere; consumer trust issues.
• Gene‑drive technology – Potential to suppress disease‑vector mosquitoes; raises ecological and governance concerns.

Case‑study prompt (AO3): “Debate the use of gene‑drive mosquitoes to control malaria. Consider scientific effectiveness, ecological risk, ethical acceptability, and regulatory frameworks.”

9. Practical Skills & Data Handling

9.1 Designing a Cloning Experiment (AO2)

• Choose restriction enzymes that give compatible sticky ends and do not cut within the gene.
• Calculate the insert:vector molar ratio:

  moles = (ng × 6.02 × 10²³) ÷ (bp × 660 g mol⁻¹)

  Typical target: 3 µmol insert : 1 µmol vector.
• Plan controls:
  1. Vector‑only ligation (to assess background).
  2. Insert‑only ligation (to check for self‑ligation).
  3. No‑ligase control (to confirm ligase activity).

9.2 Gel Electrophoresis & Confirmation (AO2)

• Run digested DNA on 0.8–1.0 % agarose with a 1 kb ladder.
• Interpret band pattern:
  • Single band at expected size → successful digestion.
  • Smear or additional bands → incomplete digestion or star activity.
• Colony‑PCR: 30‑cycle PCR using vector‑forward and insert‑reverse primers; analyse product size on agarose gel.
• Final verification by restriction mapping and Sanger sequencing.

9.3 Evaluating Experimental Errors (AO3)

10. Linkages to Other Syllabus Areas

• DNA replication – Understanding of template vs. coding strands is essential for primer design and restriction‑site placement.
• Protein synthesis – Knowledge of transcriptional and translational control informs promoter and RBS selection.
• Mutation & evolution – Site‑directed mutagenesis (a recombinant technique) allows investigation of specific nucleotide changes on protein function.
• Selection & evolution – Transgenic model organisms (e.g., GFP‑expressing Drosophila) are used to study evolutionary concepts such as fitness and adaptation.
• Biotechnology & industry – AO1 “Describe” and AO2 “Explain” are addressed when students justify the choice of vector, host, and expression system for a given product.