Gene Technologies and Biotechnology

Recombinant DNA is DNA that contains genes from two or more different organisms.

Key Tools

Making Recombinant DNA

1. Isolate the gene of interest:
   • Use restriction enzymes to cut it from donor DNA, OR
   • Use reverse transcriptase to make cDNA from mRNA, OR
   • Use a gene machine (automated DNA synthesiser) to make the gene artificially
2. Cut the vector (e.g., plasmid) with the same restriction enzyme → produces complementary sticky ends
3. Mix the gene with the cut vector → sticky ends base-pair by complementary base pairing
4. DNA ligase seals the joins → recombinant plasmid formed
5. Transform host cells (e.g., bacteria) with the recombinant plasmid
   • Methods: heat shock, electroporation, calcium chloride treatment
6. Select transformed cells using marker genes (e.g., antibiotic resistance, fluorescence)

Example: Insulin Production

• Human insulin gene is inserted into a bacterial plasmid
• Transformed bacteria are grown in fermenters
• Bacteria express the human insulin gene and produce human insulin
• Insulin is purified and used to treat diabetes
• Advantages over animal insulin: identical to human insulin (fewer allergic reactions), produced in large quantities, no animal slaughter

PCR amplifies (copies) specific DNA sequences to produce millions of copies from a tiny sample.

Requirements

• DNA template — the sample to be copied
• Primers — short single-stranded DNA sequences complementary to the flanking regions of the target DNA
• Taq DNA polymerase — a heat-stable DNA polymerase from Thermus aquaticus (a thermophilic bacterium)
• Free DNA nucleotides (dNTPs)

The PCR Cycle (3 steps, repeated 25–35 times)

1. Denaturation (~95°C, 30 sec): Heat separates the two DNA strands (breaks hydrogen bonds)
2. Annealing (~55–65°C, 30 sec): Temperature is lowered so primers bind to complementary sequences on each strand
3. Extension (~72°C, varies): Taq polymerase extends the primers, synthesising new complementary strands (5' → 3')

After $n$ cycles: $2^n$ copies of the target DNA

• After 30 cycles: $2^{30} \approx 1 \text{ billion copies}$

Applications of PCR

• Forensic science — amplify DNA from tiny samples (crime scenes)
• Medical diagnosis — detect pathogens (e.g., COVID-19 PCR tests)
• Genetic testing — screen for genetic disorders
• Research — study genes from ancient or scarce samples
• Paternity testing

Gel electrophoresis separates DNA fragments by size.

Procedure

1. DNA samples (often cut with restriction enzymes or amplified by PCR) are loaded into wells at one end of an agarose gel
2. An electric current is applied: DNA is negatively charged (due to phosphate groups) and moves towards the positive electrode (anode)
3. Smaller fragments move faster and further through the gel
4. Larger fragments move more slowly (more resistance from the gel matrix)
5. After separation, DNA is visualised:
   • Staining with ethidium bromide (fluoresces under UV light)
   • Or using a DNA probe (radioactive or fluorescent label)

Applications

• Genetic fingerprinting (DNA profiling) — comparing DNA banding patterns
• Checking the size of PCR products or restriction fragments
• Comparing DNA from different organisms

Genetic fingerprinting produces a unique banding pattern for each individual (except identical twins).

How It Works

1. Extract DNA from a sample (blood, hair, saliva)
2. Amplify the DNA using PCR (if the sample is small)
3. Cut the DNA with restriction enzymes — cutting at specific sites produces fragments of different sizes
4. Separate fragments by gel electrophoresis
5. Transfer the pattern to a nylon membrane (Southern blotting)
6. Hybridise with radioactive or fluorescent DNA probes that bind to specific variable number tandem repeats (VNTRs) or short tandem repeats (STRs)
7. Visualise the banding pattern using X-ray film or UV light

Applications

• Forensics: Matching suspects to crime scene evidence
• Paternity/maternity testing: Children inherit bands from both parents
• Immigration cases: Verifying family relationships
• Conservation: Identifying species and genetic diversity

Gene therapy involves correcting a genetic disorder by introducing a functional copy of the faulty gene into a patient's cells.

Types

• Somatic cell gene therapy: Corrects genes in body cells (not passed to offspring). Must be repeated as cells are replaced.
• Germ line gene therapy: Corrects genes in gametes or embryos (heritable — changes passed to offspring). Currently banned in humans due to ethical concerns.

Methods of Delivery (Vectors)

• Viruses (adenoviruses, retroviruses, lentiviruses) — modified to carry the therapeutic gene without causing disease
• Liposomes — lipid vesicles that fuse with cell membranes
• Direct injection of DNA

Example: Cystic Fibrosis

• CF is caused by a mutation in the CFTR gene (chloride channel protein)
• Gene therapy aims to deliver a functional CFTR gene to lung epithelial cells
• Challenges: gene expression is temporary; immune response to vectors; difficulty reaching all affected cells; viral vectors may integrate randomly

Limitations

• Effects may be temporary (need repeated treatments)
• Immune response against the vector
• Risk of insertional mutagenesis (gene inserts into the wrong place, potentially disrupting tumour suppressor genes)
• Difficult to target all affected cells

CRISPR-Cas9 is a revolutionary gene-editing tool that allows precise modifications to DNA.

How It Works

1. A guide RNA (gRNA) is designed to be complementary to the target DNA sequence
2. The gRNA directs the Cas9 enzyme (a molecular "scissors") to the exact location in the genome
3. Cas9 makes a double-strand break in the DNA at the target site
4. The cell's repair mechanisms then either:
   • Disrupt the gene (insertions/deletions from error-prone repair — NHEJ) → gene is "knocked out"
   • Insert a new sequence (if a DNA template is provided — homology-directed repair) → gene is corrected or replaced

Applications

• Correcting genetic diseases (sickle cell, CF, muscular dystrophy)
• Creating disease-resistant crops
• Studying gene function (knock out genes to observe effects)
• Cancer research and potential treatments
• Pathogen control (gene drives to modify mosquito populations)

Ethical Considerations

• Editing germ line cells (heritable changes) raises concerns about "designer babies" and unintended consequences for future generations
• Off-target effects — Cas9 might cut the wrong part of the genome
• Accessibility — expensive technology may increase health inequalities
• Ecological concerns — gene drives could have unpredictable effects on wild populations

Question: Describe the three stages of the polymerase chain reaction and explain why Taq polymerase is used. (5 marks)

Solution:

In denaturation (~95°C), the DNA sample is heated to break the hydrogen bonds between complementary base pairs, separating the double-stranded DNA into two single strands. In annealing (~55–65°C), the temperature is lowered so that short DNA primers can bind (anneal) to their complementary sequences flanking the target region on each strand. In extension (~72°C), Taq DNA polymerase adds free nucleotides to the primers, synthesising new complementary strands in the 5'→3' direction.

Taq polymerase is used because it is isolated from a thermophilic bacterium (Thermus aquaticus) and is heat-stable — it does not denature at 95°C. Normal DNA polymerase would be destroyed by the high temperatures required for denaturation, so Taq polymerase can remain active through many repeated cycles.

• Recombinant DNA is made using restriction enzymes (cut) + DNA ligase (join) + vectors (transfer).
• PCR amplifies DNA: denaturation → annealing → extension (using heat-stable Taq polymerase); $2^n$ copies after $n$ cycles.
• Gel electrophoresis separates DNA by size (smaller = further); used in genetic fingerprinting.
• Gene therapy introduces functional genes to treat genetic disorders; somatic (non-heritable) vs germ line (heritable).
• CRISPR-Cas9 enables precise gene editing using guide RNA and Cas9 nuclease; enormous potential but raises ethical concerns.