Biotechnology & Virology – Comprehensive Study Notes

Biotechnology Fundamentals

• Definition: Any technology that uses living systems, organisms, or derivatives to make or modify products/processes.

• San Diego highlighted as a biotech hub (gene therapy, diagnostic kits, creation of novel clones).

• Raw material = DNA. Manipulating, mutating, or studying DNA underpins every downstream technique.

• Thermal denaturation of DNA: strands separate just below boiling (≈ 90\,^{\circ}\text{C}) allowing access to nucleotides for identification, replication & transcription.

DNA Manipulation Tools

• Restriction Endonucleases

  • Enzymes that cut DNA cross-wise.

  • Can be “programmed” to seek a specific sequence—analogous to using AI for literature searches. Saves manual scanning of long genomes.

  • Recognize palindromic sequences—identical when read 5' \to 3' on one strand and 5' \to 3' on the complementary strand (reverse direction).

• DNA Ligase

  • Re-joins phosphate backbones, resealing breaks created by restriction enzymes.

• Circular DNA

  • Found in bacteria (e.g. plasmids in E. coli).

  • Classic cloning demonstrations: insert fluorescent-protein genes → descendants glow, proving successful gene transfer.

PCR and Electrophoresis

• Polymerase Chain Reaction (PCR) = molecular Xerox.

  1. Denaturation – heat opens double helix.

  2. Priming – add oligonucleotide primers that anneal to exposed strands.

  3. Extension – DNA polymerase builds new strands, exponentially amplifying target.

• Sample workflow example:

  • Excise mouse-tail tissue → digest with proteolytic enzymes → centrifuge → collect lysate containing pure DNA → subject to PCR cycles.

• Electrophoresis Gel

  • DNA is negatively charged (phosphate backbone).

  • Migration occurs from negative cathode → positive anode through agarose/polyacrylamide gel.

  • Buffer often 10 % Tris solution.

  • Real-time systems allow visualization of bands as they separate.

Recombinant DNA & Cloning

• "Recombinant DNA" = cloning: remove genetic material from donor A, combine with host B, offspring inherit blended genome.

Bacteriophage Therapy

• Bacteriophage (phage): viruses that infect bacteria only → harmless to humans.

• Clinical use case (story):

  • Severe leg wound → resistant infection → IV antibiotics fail → risk of amputation/sepsis.

  • Solution: manufacture phage that target the specific bacterial strain, inject, and clear infection.

• Challenges: custom production, high cost, available only in specialized biotech facilities.

DNA Size & Genomics

• Approximate base-pair counts mentioned:

  • E. coli plasmid example: 1{,}300\ \text{bp} (illustrative).

  • Human mitochondrial genome: 16\ \text{kilo-bases (kb)}.

  • Human nuclear genome: 6\times10^{9}\ \text{bp}.

• Human Genome Project

  • Duration: 13\ \text{years}.

  • Today: comparable sequencing accomplished overnight on supercomputers.

Biotechnological Products in Medicine

• Interferons – antiviral & anticancer peptides (treat cancer, MS, hepatitis, genital warts).

• Interleukins (IL) – cytokines; hundreds exist. Key examples:

• trigger fever/inflammation.

  • \text{IL-10} → initiates inflammation modulation.

  • Research in Florida: mRNA platform instructing cells to overexpress TNF-α & ILs to attack all cancers.

• Humira (adalimumab) – monoclonal antibody (mAb) for autoimmune conditions (Crohn’s, RA).

• Erythropoietin (EPO) – kidney-derived hormone; stimulates RBC production. Needed in renal failure & dialysis.

• Human Growth Hormone (hGH) – treats dwarfism, prevents wasting, also abused for physique enhancement.

• Recombinant human DNase I (dornase alfa) – breaks viscous mucous in cystic fibrosis (CF)

  • CF: mutated \text{CFTR} gene on chromosome 7, defective Na⁺/Cl⁻ channel → thick secretions in lungs, GI, reproductive tracts.

• Tissue Plasminogen Activator (tPA)

  • Clot-buster for acute stroke & myocardial infarction.

  • 100\,\text{mL} vial ≈ \$5{,}000; usual dose 80\,\text{mL} (20 mL discarded).

  • Therapeutic window: <4\ \text{hours} from symptom onset.

  • Only ≈30\ \% efficacy in ischemic stroke; risk of hemorrhage.

Vaccine Production (Recombinant)

• Non-pathogenic subunits engineered via recombinant DNA.

• Examples:

  • HPV (gardasil types 6, 8, 11, etc.) – prevents cervical cancer & warts.

  • Hepatitis B – protects liver; "hepatitis" = inflammation of liver.

  • Haemophilus influenzae type B (HiB) – prevents pediatric meningitis & epiglottitis.

Virology Basics

• Virus architecture

  • Envelope replaces cell wall; beneath lies capsid enclosing nucleic acid.

  • Genomes are either DNA or RNA (never both).

• Replication sites

  • DNA viruses → nucleus.

  • RNA viruses → cytoplasm.

• Retrovirus (HIV)

  • Carries reverse transcriptase → converts RNA → DNA ("replicates backwards").

Bacteria vs Viruses & Microbial Structures

• Bacteria

  • No true nucleus; possess circular DNA (plasmids).

  • Gram-Negative more virulent than Gram-Positive:

  • Presence of lipopolysaccharide (LPS) endotoxin.

  • Evades lysosomal destruction.

  • Endospores: vulnerable only during growth/sporangium phase; high heat can destroy.

  • Mycobacterium & Nocardia

  • Acid-fast cell wall with mycolic acid (waxy) → resists phagocytosis.

  • Cells often pleomorphic (variable shape).

• Viruses

  • Considered non-living (lack metabolic machinery).

  • Function as obligate intracellular parasites—classic parasitic definition (take but give nothing).

• Prions

  • Infectious proteins; extremely resistant to conventional sterilization.

Antimicrobials: Antibiotics vs Antivirals

• Antibiotics

  • Target bacteria directly:

  • \text{Bactericidal} – destroy cell wall, e.g.
    \beta-lactams.

  • \text{Bacteriostatic} – inhibit functions like 70\,\text{S} ribosome.

  • Fewer host side effects because human cells lack target structures.

• Antivirals

  • Inhibit virus life-cycle within host cell (entry, transcription, assembly).

  • Inevitably interfere with host processes → higher side-effect profile (immune suppression, cytotoxicity, etc.).

Insulin Case Study

• Historical supply: Extracted from cows, pigs, horses (first trials: dog).

• Modern supply: Mass-produced via recombinant DNA in microbes → identical to human insulin, unlimited quantities.

Miscellaneous Concepts & Quick Facts

• Electrophoresis buffer: "10 % Tris" for conductivity without causing user shock.

• Cystic Fibrosis therapy: RhDNase breaks mucus; patients still infertile due to reproductive tract plugging.

• TPA dosage quirks: Manufacturer vial & dosing mismatch → routine wastage.

• Potential biotech careers: Pharmacology research, clinical trials, lab design—high earning potential.

Study Strategy (Instructor Advice)

• Re-read the standardized blueprint plus these notes.

• Focus on conceptual groupings (e.g.

  • Cell wall structures

  • DNA manipulation enzymes

  • Biotech-derived drugs & their targets).

• Expect application-level questions rather than verbatim repeats from practice sets.