Biotechnology Overview: rDNA, Cloning, PCR, and Antibodies

Introduction to Recombinant DNA, Cloning & PCR

Objectives

• Understand key terms:
  • Gene
  • Cloning
  • Polymerase Chain Reaction (PCR)
• Examine steps of cloning.
• Describe roles of enzymes in cloning:
  • Restriction endonucleases
  • Ligases
• Explore plasmids and their roles:
  • Autonomous replication
• Discuss expression systems used in cloning.
• Outline PCR process steps.

Principles of Genomes

• Definition of Genome: Complete set of genes or genetic material in a cell.
• Genomics: Study of all DNA, including genes, relevant for biological research.
• Importance: Provides key information for biological processes and can be manipulated to alter functions.

Genes Defined

• Basic unit of inheritance; specify traits passed from parents to offspring.
• Human genome: Approx. 20,000 protein-coding genes, <1.5% of DNA is coding (exons).
  • Introns: Non-coding regions of DNA.
  • Exons: Coding segments.

Gene Expression Process

1. Transcription: DNA -> pre-mRNA -> mRNA (RNA splicing removes introns).
2. Translation: mRNA -> Protein (requires ribosomes and tRNA for amino acids).

Cloning Process Overview

• DNA Cloning: Amplification of specific DNA segments for study.
• Steps in DNA Cloning:
  1. Obtain DNA segment.
  2. Select cloning vector (small DNA molecule).
  3. Covalently join DNA fragments.
  4. Introduce recombinant DNA into host.
  5. Identify host cells with recombinant DNA.

Cloning Vectors

• Cloning vectors (e.g., plasmids) can replicate autonomously and include:
  • Bacterial artificial chromosomes (BACs)
  • Yeast artificial chromosomes (YACs)
• Example - pBR322: Common synthetic plasmid with features for cloning.

Restriction Endonucleases

• Function: Recognize and cleave DNA at specific sequences, resulting in sticky or blunt ends.
• Types:
  • Type I and III: Complex, ATP-dependent enzymes.
  • Type II: Simpler, hydrolytic cleavage and palindromic recognition sequences (e.g., BamHI, HindIII).

DNA Ligases

• Connect two DNA fragments, facilitating the final step of DNA cloning.

Expressing Cloned Genes

• Expression Vectors: Contain transcription and translation signals for gene regulation (e.g., E. coli systems).
• Benefits include high yield and specific activity of proteins produced.

Introduction to PCR

• Definition: Amplification of a DNA segment to millions of copies within hours.
• Uses:
  • Sequencing DNA
  • Forensic analysis
  • Genetic engineering

PCR Process Steps

1. Denaturation: Heat causes DNA separation.
2. Annealing: Cool to allow primers to attach to the target sequence.
3. Extension: Taq polymerase synthesizes new DNA strands.

Reverse Transcriptase PCR (RT-PCR) and qPCR

• Converts RNA into complementary DNA (cDNA) before amplification.
• qPCR: Quantitative method, uses fluorescent dyes to track DNA concentration.

Introduction to Monoclonal Antibodies

Objectives

• Understand antibody structures and functions.
• Differentiate between monoclonal and polyclonal antibodies, including their varying forms (murine, chimeric).

Antibody Structure

• IgG: Most common, consists of heavy and light chains with distinct antigen binding (Fab) and biological activity (Fc) portions.
• Classes of antibodies:
  • IgG, IgM, IgA, IgD, IgE with various roles in immune response.

Monoclonal vs. Polyclonal Antibodies

• Monoclonal Antibodies: Homogeneous, produced from a single clone (specific epitope binding).
• Polyclonal Antibodies: Heterogeneous mix, multiple clones (varied epitope binding).

Applications of Monoclonal Antibodies

• Treatment for:
  • Cancer
  • Autoimmune disorders
  • COVID-19
  • Other diseases in need of targeted therapy.

Production and Nomenclature of mAbs

Production Methods:

• Hybridoma technology for producing stable mAbs.
• Transgenic mice with humanized antibodies.

Protein Drugs

• Significance: Key category of pharmaceuticals due to their role in various biological actions and minimal side effects compared to small molecules.

Protein Stability Challenges

• Instability due to aggregation, chemical modifications, and denaturation.
• Lyophilization: A method to enhance protein drug stability through freeze-drying processes.

Lyophilization Stages:

1. Freezing: Solidify the product, preventing interactions.
2. Primary Drying: Remove ice via sublimation.
3. Secondary Drying: Desorb moisture remaining in matrix to enhance shelf-life.