Objective:
This practical module provides hands-on training in fundamental molecular biology techniques, focusing on DNA extraction, quantification, amplification, and analysis. It equips students with the skills to isolate and characterize genetic material from diverse biological sources.
1. Core Techniques Covered
DNA Extraction: Methods for isolating genomic DNA from animal (rat blood) and plant (strawberry leaf) tissues using chemical lysis and precipitation.
DNA Quantification & Purity Assessment: Spectrophotometric analysis (A260/A280 and A260/A230 ratios) to determine concentration and purity.
DNA Melting Temperature (Tm): Measuring Tm to estimate GC content and DNA stability.
Agarose Gel Electrophoresis: Separating DNA fragments by size to assess integrity and confirm amplification.
Polymerase Chain Reaction (PCR): Amplifying specific DNA sequences using primers, thermostable polymerase, and thermal cycling.
2. Key Experiments (TP)
TP1: DNA Extraction from Rat Blood
Goal: Isolate genomic DNA using red/white blood cell lysis, protein precipitation, and ethanol purification.
Techniques: Centrifugation, chemical lysis, chloroform extraction, DNA spooling.TP2: DNA Extraction from Plant Tissue
Goal: Isolate DNA from strawberries using mechanical disruption (grinding) and detergent-based lysis.
Techniques: Mortar/pestle grinding, filtration, ethanol precipitation.TP3: DNA Characterization
Goal: Determine DNA concentration, purity (spectrophotometry), and GC content (Tm measurement).
Techniques: UV spectrophotometry, melting curve analysis.TP4: Agarose Gel Electrophoresis
Goal: Visualize DNA fragments, assess integrity, and estimate size using molecular weight markers.
Techniques: Gel casting, sample loading, ethidium bromide staining, UV visualization.TP5 & TP6: PCR and Optimization
Goal: Amplify target DNA sequences and optimize annealing temperature for specificity.
Techniques: Primer design, master mix preparation, thermal cycling, gradient PCR.
3. Theoretical Concepts Applied
DNA Structure & Stability: Understanding hyperchromicity, Tm, and GC content.
PCR Principles: Primer design (length, GC%, Tm, specificity), reaction components (template, primers, dNTPs, Taq polymerase), and cycling conditions.
Electrophoresis: Separation based on size, charge, and agarose concentration.
Safety & Ethics: Handling mutagens (e.g., ethidium bromide), biological hazards, and recombinant DNA.
4. Applications
Genetic Research: Gene cloning, mutation detection, genotyping.
Medical Diagnostics: Pathogen detection, genetic disorder screening.
Forensics & Agriculture: DNA fingerprinting, GMO analysis, crop improvement.
Biotechnology: Recombinant protein production, synthetic biology.
Conclusion:
This module bridges theory and practice, enabling students to master essential molecular biology techniques. Skills gained here are critical for careers in research, diagnostics, and biotech industries, emphasizing precision, safety, and analytical rigor.
- Enseignant: IDIR MOUALEK