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Molecular Biology & Medical Biotechnology Exam Notes

DNA Orientation & Circular Genomes

  • DNA strands are antiparallel.
    • One strand is read 5' \rightarrow 3', the complementary strand 3' \rightarrow 5'.
  • Circular DNA:
    • Found primarily in bacteria (e.g.
      E.\ coli plasmids) and some organelles.
    • Enables cloning: foreign genes can be inserted, replicated, and expressed.

Key Enzymes in DNA Manipulation

  • Endonuclease (restriction enzyme)
    • Cuts DNA at specific palindromic sequences ("sticky" or "blunt" ends)
    • Essential for excising or inserting genes.
  • Ligase
    • Reseals phosphodiester backbone after insertion/removal.
    • "Molecular glue" in recombinant work.

Retroviruses & Reverse Transcriptase

  • HIV is a retrovirus because it performs reverse transcription:
    • Converts viral RNA \rightarrow complementary DNA (cDNA).
    • Enzyme: Reverse Transcriptase (target of many antiretrovirals).

Polymerase Chain Reaction (PCR)

  • Purpose: exponential amplification of a chosen DNA segment (molecular Xerox).
  • Core steps repeated 25–35×:
    1. Denaturation (~95 °C): double helix opens.
    2. Annealing/Priming (~50–65 °C): short oligonucleotide primers bind.
    3. Extension (~72 °C): thermostable DNA polymerase adds dNTPs.
  • Primers can carry tags, mutations, or restriction sites for downstream cloning.
  • Millions of copies generated in hours from minimal starting template.

Gel Electrophoresis

  • Agarose/poly-acrylamide matrix + electric field separates DNA by size.
    • DNA migrates toward the anode (positive pole) because its phosphate backbone is negative.
  • Produces a ladder pattern; band intensity reflects quantity.
  • Critical for verifying PCR/cloning products before ligation or sequencing.

Bacteriophage Therapy

  • Bacteriophages: viruses that exclusively infect bacteria; harmless to humans.
  • Conceptual clinical rescue scenario:
    • Antibiotic-resistant limb infection \rightarrow programmed phage cocktail \rightarrow lysis of culprit bacteria without amputation.
  • Barriers: cost, customization time, regulatory logistics.

Genome Sizes & Sequencing Milestones

  • Representative sizes:
    • E.\ coli \approx 1\,300 base pairs in example plasmid context.
    • Human mitochondrial genome \approx 16 kilobases (kb).
    • Human nuclear genome \approx 6\times10^{9} base pairs.
  • Human Genome Project (early 2000s):
    • Initial draft: 13 y, costs \$3\,000\,000\,000.
    • Today: full genome <\$1\,000, completed in one afternoon by high-throughput sequencers.

Recombinant DNA–Derived Biopharmaceuticals

Interferons

  • Antiviral, anti-tumor, and immunomodulatory peptides.
  • Treat: hepatitis B/C, multiple sclerosis, some cancers, genital warts.

Interleukins (IL) & Tumor-Necrosis Factor-α (TNF-α)

  • Cytokines that amplify immune surveillance against tumors.
    • IL-1 & IL-6: fever induction.
    • IL-10: inflammation modulation.
  • mRNA-based platforms now explored to up-regulate TNF-α/ILs for tumor destruction.
  • “-mab” drugs (monoclonal antibodies; e.g.
    Humira) target cytokine pathways in autoimmune diseases (RA, Crohn’s, etc.).

Hematopoietic & Growth Factors

  • EPO (erythropoietin):
    • Produced natively by kidneys; stimulates bone-marrow erythrocyte synthesis.
    • Dialysis patients require recombinant EPO injections.
  • Human Growth Hormone (hGH): pediatric growth failure, cachexia, performance enhancement (illicit use).

Recombinant Enzymes & Misc.

  • Rh\ DNase (Pulmozyme): liquefies mucus in cystic fibrosis.
    • Targets mutated CFTR \Rightarrow thick secretions.
  • tPA (tissue plasminogen activator): dissolves clots in acute ischemic stroke & myocardial infarction.

Vaccines via Recombinant DNA

  • Pathogenic genes removed; immunogenic epitopes retained.
  • Examples: Hepatitis B surface antigen, HPV L1 capsid protein.

Gene Therapy

  • Vectors (adenovirus, lentivirus) deliver corrective genes.
  • Goal: halt or slow genetic disorders rather than full cure.
  • FDA-approved cases exist; pipeline expanding.

Fundamental Virology

  • Virion components:
    • Envelope (lipid) ± capsid (protein) housing nucleic acid.
  • Replication site depends on genome type:
    • DNA viruses: nucleus.
    • RNA viruses: cytoplasm.
  • Viruses lack metabolism; parasitize host machinery ⟹ “obligate intracellular parasites.”

Bacteria vs. Eukaryotes

  • Bacteria: no nucleus, circular DNA, 70 S ribosomes, cell wall with peptidoglycan.
  • Eukaryotes (humans): nucleus, linear chromosomes, 80 S ribosomes, sterol-rich membrane.

Insulin Production

  • Prior source: porcine/bovine pancreata (impurities, immunogenic).
  • rDNA breakthrough enabled large-scale human insulin synthesis in E.\ coli laboratories.

Prions

  • Misfolded, infectious proteins (no nucleic acid).
  • More resistant than bacterial endospores to heat, chemicals, radiation.

Antivirals vs. Antibiotics

  • Antibiotics: target bacterial structures absent in humans (peptidoglycan, 70 S ribosome) → fewer side-effects.
  • Antivirals: must inhibit host cell processes co-opted by viruses → higher toxicity profile.

Cell Wall Complexity & Gram Status

  • Gram-positive: thick peptidoglycan, teichoic acids.
  • Gram-negative: thin wall + outer LPS layer.
    • LPS (lipopolysaccharide) = endotoxic, major virulence factor.
    • Therefore, gram-negative infections often more dangerous.

Special Bacterial Structures

  • Mycobacterium & Nocardia: mycolic-acid-rich, waxy outer layer → acid-fast, pleomorphic shapes.
  • Pleomorphism: ability to vary morphology (e.g.
    coccobacilli).

Fungi & Parasites (Quick Review)

  • Fungi possess chitinous cell walls (distinguishing from plants & animals).
  • Parasite definition: organism that benefits at host’s expense.
    • Viruses considered the closest microbial analogue to obligate parasites.

Essential Vocabulary Refresher

  • Endonuclease: sequence-specific DNA scissors.
  • Ligase: phosphodiester glue.
  • Electrophoresis: charge-driven size separation technique.
  • Bacteriophage: bacteria-specific virus.
  • PCR Primer: short synthetic oligonucleotide guiding DNA polymerase.
  • Reverse Transcriptase: RNA-dependent DNA polymerase unique to retroviruses.