Notes on DNA Extraction and Applications in Molecular Biology

INTRODUCTION

  • Overview of DNA extraction process: removal of DNA from other cellular components.

STEPS IN DNA EXTRACTION

STEP 1: LYSIS

  • Definition: The process of breaking down the cell to release DNA from the nucleus.
  • Alternatives: Known as cell lysis or cell disruption.
  • Methods:
    1. Physical method:
    • Targets cells with strong membranes like plant cells.
    • Techniques include:
      • Blender
      • Mortar and pestle (ครกและสาก)
      • Cutting (การตัด)
      • Sonicator (คลื่นเสียงความถี่สูง)
    1. Chemical method:
    • Destroys nuclear membrane (nuclear membrane lysis).
    • Uses detergents, e.g.:
      • Sodium dodecyl sulfate (SDS)
      • Lauryl sulfate
    • Employs enzymes to break bonds in the proteins, e.g.:
      • Proteinase K
      • Cellulase
      • Lyticase
      • Lysozyme

STEP 2: PRECIPITATION

  • Definition: The process of separating DNA from cellular debris after lysis.
  • Post-lysis content:
    • Cell extract containing DNA + proteins + detergents + other chemicals.
  • Mechanism:
    • Sodium ions (Na⁺) neutralize the charges of DNA, stabilizing it.
    • Alcohol (e.g., isopropanol or ethanol) causes DNA to precipitate as it is insoluble in alcohol.
    • Proteases digest proteins that are bound to DNA.
    • Filtration aids in separating contaminants.

STEP 3: WASH / PURIFICATION

  • Process: The precipitated DNA is washed with cold alcohol.
  • Centrifugation: Used to remove contaminants such as:
    • Proteins
    • Salts

STEP 4: RESUSPENSION

  • Definition: The step where DNA pellet is dissolved in a buffer solution.
  • Common buffers used:
    • Tris buffer
    • TE buffer
    • Double distilled water
  • Outcome: DNA is ready for subsequent applications.

QUANTIFICATION OF DNA EXTRACTION

1. UV ABSORBANCE

  • Method: Measures absorbance of DNA at specific wavelengths.
  • Wavelengths:
    • DNA and RNA absorb at 260 nm.
    • Proteins absorb at 280 nm.
  • Quality indicators:
    • Ratio A260/A280 to determine purity of DNA (ideal = 1.8–2.0).
    • Ratio A260/A230 indicates contamination (ideal > 1.5).
  • Advantages:
    • Quick and does not involve chemicals.
  • Limitations:
    • Low sensitivity when DNA concentrations are low.
    • Cannot separate DNA from RNA.

2. FLUORESCENT DYES

  • Function: Used to bind specifically to double-stranded DNA (dsDNA) with examples like:
    • SYBR Green
    • PicoGreen
  • Features:
    • Higher sensitivity compared to UV absorbance.
    • Requires a standard curve for quantification.

3. AGAROSE GEL ELECTROPHORESIS

  • Purpose: Evaluates quantity and integrity of DNA across various sizes.
  • Process:
    • DNA is run on an agarose gel stained with an intercalating dye such as ethidium bromide.
  • Analysis:
    • Band intensity compared against a DNA ladder.
  • Benefits:
    • Visualizes DNA size and integrity; detects contamination.
  • Limitations:
    • Cannot quantify protein or salt contamination.

4. CAPILLARY ELECTROPHORESIS

  • Overview: Similar to agarose gel electrophoresis but in an automated system.
  • Requirements:
    • Only 1–2 µL of sample used.
    • Uses fluorescent dyes for visualization.

5. DIPHENYLAMINE METHOD

  • Reaction: Diphenylamine reacts with deoxyribose under acidic conditions.
  • Result: Produces a blue compound measurable at 595 nm.
  • Limitations:
    • Low sensitivity, time-consuming, and not commonly used now.

6. qPCR / RT-PCR

  • Function: Quantifies DNA during amplification using fluorescence.
  • Standard curve: Necessary for accurate quantification.
  • Advantages:
    • High sensitivity and accuracy.

SUMMARY

  • DNA extraction is a fundamental process in medical, biomedical science, and biotechnology.
  • Main Steps: lysis → precipitation → purification → elution.
  • Method selection depends on sample type, age, and amount.

APPLICATIONS OF MOLECULAR BIOLOGY TECHNIQUES IN MEDICINE

A. GENETIC DISEASE AND DISEASE IDENTIFICATION

  • Significance of molecular genetic testing:
    • Early detection
    • Predictive testing
    • Carrier screening
    • Personalized medicine
    • Disease prognosis.
  • Methods used:
    • Targeted testing
    • Gene panel
    • Whole-exome sequencing (WES)
    • Whole-genome sequencing (WGS)
    • Chromosomal tests (e.g., FISH, CMA, karyotype)
    • Gene expression tests
    • Biochemical tests.

B. FORENSICS

  • Applications include:
    • Paternity tests
    • Victim/suspect identification.

C. PRENATAL SCREENING AND GENETIC COUNSELING

  • Focus: Genetic mutation identification, carrier screening, family history analysis.
  • Typical methods:
    • Non-invasive prenatal testing (NIPT)
    • Chorionic villus sampling (CVS)
    • Amniocentesis.

D. DNA EXTRACTION FOR INFECTIOUS DISEASE IDENTIFICATION

  • Example: Mycobacterium tuberculosis.
  • Processes involved:
    • Sample collection
    • DNA extraction
    • PCR amplification
    • Interpretation of results.

E. PHARMACOGENOMICS

  • Study of genetic variation affecting drug response leading to:
    • Personalized medicine
    • Gene expression analysis
    • Gene editing techniques such as CRISPR-Cas9
    • Next Generation Sequencing (NGS).

F. CANCER RESEARCH AND DIAGNOSTICS

  • Focus areas:
    • Cancer biology
    • Genomic profiling
    • Liquid biopsy
    • Imaging techniques (CT, MRI, PET)
    • Precision medicine.
  • Future trends to watch:
    • Immunotherapy
    • Targeted therapy
    • Artificial Intelligence (AI) in diagnostics
    • CRISPR/Cas9 for gene editing and treatment.