Biotechnology Fundamentals: Genetics, Recombinant DNA, and Therapeutics

Posted on Nov 8, 2025 in Biology

Biotechnology: Definitions and Core Concepts

Definition: Biotechnology uses living organisms or their products to solve problems or create useful items.

Historical and Modern Techniques

Historical Uses

  • Fermentation
  • Selective breeding
  • Antibiotics

Modern Techniques

  • Gene cloning
  • Genetic engineering
  • Recombinant DNA
  • CRISPR-Cas (Gene Editing)
  • Synthetic genomes

Disciplines Involved

  • Biology
  • Chemistry
  • Physics
  • Mathematics
  • Computer science and Bioinformatics
  • Engineering

Applications of Biotechnology

  • Disease-resistant crops
  • Golden rice (enhanced nutrition)
  • Environmental cleanup (bioremediation)
  • Cultured cells for protein production

Recombinant Proteins and Therapeutics

Recombinant proteins include insulin, growth hormone, monoclonal antibodies, and clotting factors used for treating diseases.

Key Recombinant Proteins and Their Applications

  • Blood Factor VIII (Clotting Factor): Used to treat hemophilia.
  • Epidermal Growth Factor: Stimulates antibody production in patients with immune system disorders.
  • Growth Hormone: Corrects pituitary deficiencies and short stature in humans; also used in cows to increase milk production.
  • Insulin: Used to treat diabetes.
  • Interferons: Used to treat cancer and viral infections.
  • Interleukins: Used to treat cancer and stimulate antibody production.
  • Monoclonal Antibodies: Used to diagnose and treat diseases such as arthritis and cancer.
  • Tissue Plasminogen Activator (tPA): Used to treat heart attacks and strokes.

Top Biotech Drugs (2016 Examples)

These drugs are used for conditions like cancer, arthritis, diabetes, and infections:

  • Humira (AbbVie): Treats arthritis and inflammatory bowel diseases.
  • Harvoni (Gilead Sciences): Cures hepatitis C.
  • Rituxan (Roche): Targets lymphoma.
  • Revlimid (Celgene): Treats multiple myeloma.
  • Avastin (Roche): Used for various cancers including colorectal and breast.
  • Herceptin (Roche): Treats breast and gastric cancer.
  • Enbrel (Amgen): Used for arthritis and psoriasis.
  • Prevnar 13 (Pfizer): Vaccine for pneumococcal infections.
  • Lantus (Sanofi): Insulin for diabetes.
  • Neulasta (Amgen): Treats low white blood cell count (neutropenia).

Genetics, Diagnostics, and Personalized Medicine

Key Concepts in Human Genetics

  • Human Proteome: The complete set of proteins active in human cells.
  • Genetic Variation: Differences in DNA sequences among individuals.
  • SNPs (Single Nucleotide Polymorphisms): Tiny DNA mutations that vary between individuals and can cause or increase risk for diseases like:
    • Sickle cell anemia
    • Breast cancer (BRCA1/BRCA2)
    • Stroke, arthritis, diabetes, heart disease

Diagnostic Tools

  • Microarray (Gene Chip): Detects many SNPs at once to analyze a patient’s genetic profile.

Personalized Medicine Approaches

  • Pharmacogenomics: Tailors drug therapy based on individual genetic makeup.
  • Metabolomics: Measures small molecules (glucose, ATP, cholesterol) to distinguish disease from normal function.

Advanced Technologies and Industry

Advanced Technologies

  • Nanotechnology: Uses microscopic particles to deliver drugs directly to cells.
  • Gene Therapy: Replaces or augments defective genes; challenges include delivery and long-term safety.
  • Stem Cell Technology:
    • ESCs (Embryonic Stem Cells): Can become any cell type; controversial due to embryo use.
    • ASCs (Adult Stem Cells): Used for tissue repair and organ regeneration.
  • Regenerative Medicine: Genetically modifies stem cells to treat genetic disorders by reinserting corrected cells into the patient.

Ethical Issues

GMOs (Genetically Modified Organisms) and gene therapy raise public concerns and regulatory scrutiny.

Industry Comparison

  • Pharmaceutical Companies: Primarily use chemical synthesis for drug development.
  • Biotech Companies: Use living organisms to develop drugs and other products.

Molecular Biology and Cellular Foundations

Key Cellular Structures and Functions

  • Plasma Membrane: Controls entry/exit, communication, and cell shape.
  • Cytosol: Gel-like fluid inside the cytoplasm.
  • Peroxisomes: Detoxify harmful substances.
  • Centrioles: Organize cell division.
  • Cilia/Flagella: Facilitate movement.
  • Chloroplasts (plants): Site of photosynthesis.
  • Central Vacuole (plants): Used for storage.

Gene Definition and Chromosome Basics

Phosphodiester bonds link nucleotides. A gene is any DNA sequence used to produce RNA. Not all genes make proteins; genes can code for tRNA, rRNA, or regulatory RNAs. Genes influence traits and cell function.

Chromosome Structure and Organization

  • Chromatin: DNA + histone proteins; the loose form of DNA in non-dividing cells.
  • Chromosomes: Highly coiled chromatin during cell division.

Humans have 46 chromosomes (23 pairs):

  • Autosomes: Pairs 1–22.
  • Sex Chromosomes: Pair 23 (XX or XY).
  • Gametes: Haploid (23 chromosomes).
  • Somatic Cells: Diploid (46 chromosomes).

Detailed Chromosome Structure

  • Centromere: Region joining chromatids; defines the p-arm and q-arm.
  • Telomeres: Repetitive sequences at chromosome ends; protect DNA during division but shorten with age.

Karyotyping and Diagnostics

  • Karyotype: Visual chromosome map used to detect abnormalities (e.g., Trisomy 21).
  • Spectral Karyotyping: Uses fluorescent probes (e.g., FISH) to identify specific genes or changes.

The Central Dogma: Replication, Transcription, and Translation

The Central Dogma describes the flow of genetic information: DNA → RNA → Protein.

DNA Replication Steps

  1. Unwinding: Helicase unzips DNA; single-strand binding proteins stabilize the strands.
  2. Priming: Primase adds RNA primers.
  3. Copying: DNA polymerase builds the new strand (5’ to 3’).
  4. Lagging Strand: Discontinuous synthesis via Okazaki fragments.
  5. Finishing: DNA ligase seals the fragments.

Transcription (DNA to RNA)

  • RNA polymerase binds to the promoter and copies DNA into RNA.
  • The template strand is read 3’ to 5’; RNA is built 5’ to 3’.
  • Base pairing rules: Adenine (A) pairs with Uracil (U), Cytosine (C) pairs with Guanine (G).
  • A termination sequence ends transcription.
  • mRNA (Messenger RNA): Carries the genetic code to the cytoplasm.

Translation Stages (RNA to Protein)

  1. Initiation: Ribosome binds mRNA; finds the start codon.
  2. Elongation: tRNAs bring amino acids; peptide bonds form.
  3. Termination: A stop codon triggers the release of the completed protein.

Recombinant DNA Technology and Gene Cloning

Core Tools and Concepts

  • Recombinant DNA: DNA formed by combining genetic material from two different sources.
  • Gene Cloning: Making identical copies of a specific gene or DNA segment.
  • Restriction Enzymes: Enzymes that cut DNA at specific palindromic sequences.
    • They produce either sticky ends (preferred for cloning) or blunt ends.
    • Example: EcoRI cuts at GAATTC.
  • Plasmid Vectors: Small circular DNA used to carry foreign DNA into host cells.
    • Must have: an origin of replication (ori), a selectable marker (e.g., antibiotic resistance), and a multiple cloning site (MCS).

Creating Recombinant DNA

  1. Cut the plasmid and the target DNA with the same restriction enzyme.
  2. Join the fragments using DNA ligase.
  3. Insert the recombinant plasmid into bacteria via transformation or electroporation.

Selection Methods for Recombinants

  • Antibiotic Selection: Only bacteria that successfully took up the plasmid survive.
  • Blue-White Screening:
    • Insert disrupts the lacZ gene → resulting in white colonies (recombinant).
    • Functional lacZ → resulting in blue colonies (non-recombinant).

DNA Libraries

  • Genomic Library: Contains all DNA (coding + non-coding).
    • Disadvantage: Includes introns; large and time-consuming to screen.
  • cDNA Library: Made from mRNA using reverse transcriptase.
    • Advantage: Only includes expressed genes (no introns).

Screening Libraries

Use probes (complementary DNA/RNA) to find genes of interest. Modern sequencing often replaces traditional library screening.

Polymerase Chain Reaction (PCR)

Developed by Kary Mullis, PCR amplifies specific DNA sequences rapidly.

PCR Steps

  1. Denaturation (94–96°C): DNA strands separate.
  2. Annealing (55–65°C): Primers bind to the target DNA.
  3. Extension (70–75°C): DNA polymerase synthesizes new DNA.
  • Taq Polymerase: A heat-stable enzyme from Thermus aquaticus.
  • Amplification Formula: After n cycles, the result is 2ⁿ copies.

PCR Applications

  • Gene expression studies
  • Disease diagnosis
  • Forensics and paternity testing
  • Ancient DNA analysis

Cloning PCR Products

Taq polymerase adds an extra Adenine (A) to the 3’ ends. Use T vectors with 3’ Thymine (T) overhangs for efficient ligation.