DNA Isolation Techniques and Applications 3

Isolation of Genomic DNA

• The goal is to isolate high-molecular-weight DNA, free of proteins and enzyme inhibitors.
• This purified DNA is crucial for subsequent DNA-related work.
• Applications include genetic disease detection, cancer predisposition assessment, next-generation drug production, cell therapy, and genetic engineering.

DNA Structure

• DNA is a double helix.
• The double helix is composed of a sugar-phosphate backbone.
• Nucleotides, containing bases (adenine, thymine, cytosine, and guanine), form the "rungs" of the ladder.
• Hydrogen bonds connect paired bases (A with T, and C with G).

Choosing a DNA Isolation Method

• The best method depends on the type of nucleic acid needed (genomic, mitochondrial, plasmid).
• The source material's origin (plant, animal, bacterial, viral).
• The material type (tissues, organs, cell cultures).
• The expected results (purity, quality, time).
• The intended purpose (PCR, cloning, etc.).

Materials for Isolation (Human)

• Peripheral blood is a common source, isolated using EDTA tubes.
• EDTA prevents blood clotting and inhibits deoxyribonucleases (DNases).
• Heparin-containing tubes can negatively impact subsequent stages.
• Other sources include epithelial cells, fibroblast cultures, amniotic fluid cells (AFC), chorionic villus sampling (CVS), and hair follicles.
• Less common materials include blood spots, semen, tissue fragments, and bone marrow.

Stages of DNA Isolation

• Collection of the biological material sample.
• Transport of the collected material.
• Homogenization of the collected material.
• Protein lysis (denaturation).
• Purification of DNA from lysate remnants.
• Dissolving DNA.
• Qualitative and quantitative evaluation of the isolated DNA.

Transport

• Proper labeling is essential.
• Secure sealing of tubes or containers is necessary.
• Protection from damage and contamination is crucial.
• Protection from temperature changes during transport is needed.

Storage

• Immediately begin DNA isolation after sample collection.
• Tissues can be stored at 4°C for several days without significant DNA degradation.
• If isolation isn't possible within 48 hours of collection, freeze at -20°C or -80°C.
• Longer storage reduces both yield and DNA quality.

Homogenization

• The method depends on material origin.
• Soft animal tissues: homogenization.
• Cell suspensions and biological fluids: sonication (ultrasound) or chemical lysis.
• Plant cells: mechanical grinding.
• Bacterial cells and cell cultures: chemical lysis.

Cell Lysis

• Disrupts cell membranes to release DNA/RNA.
• Denatures proteins and inactivates nucleases.
• Methods: Enzymatic (proteinase K, collagenase, trypsin, chitinase), Chemical (chaotropic salts like guanidine isothiocyanate, detergents like CTAB, SDS, Triton X-100), Physical (heating, freezing, osmotic shock), and Mechanical (glass beads, aluminum oxides).

DNA Isolation Methods

• Solvent-based: Phenol-chloroform extraction, protein precipitation.
• Chromatographic: Binding DNA to carrier material (silica columns), magnetic separation.

Purification of DNA (organic method)

• Centrifugation to separate DNA from associated compounds.
• Use of a saturated phenol solution buffer, chloroform with isoamyl alcohol, mixture of phenol with chloroform and isoamyl alcohol (25:24:1).

Purification of DNA (salt method)

• Lysis, followed by centrifugation after adding sodium chloride.
• Precipitation process.

DNA Isolation by Column Method

• DNA binds to ion-exchange membranes, removing contaminants.
• The column is washed to remove impurities.
• DNA is eluted (removed from the membrane) using a low-salt buffer.

DNA Isolation by Magnetic Method

• DNA binds to paramagnetic beads.
• Removal of contaminants using washing steps.
• DNA is eluted into solution.

Quantification and Quality Assessment

• Crucial step for genetic material work.
• Contaminants (phenol, EDTA, heparin) can negatively impact analytical techniques (especially PCR).
• Methods for measuring DNA concentration: spectrophotometry, agarose gel electrophoresis.

Spectrophotometric Measurement

• Dilute DNA/RNA samples with water or TE buffer.
• Measure absorbance at specific wavelengths:
  • 260 nm (DNA/RNA absorb strongly).
  • 280 nm (proteins absorb).
• Ratio of absorbance at 260nm/280nm (A260/A280) indicates purity, typical ratios are:
  • dsDNA ~1.8.
  • RNA ~2.0.
  • Values below 1.8 suggest protein contamination, 1.5 ≈ 50% contamination.