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• Reasons to isolate nucleic acids:

  • Obtain high-quality DNA for various applications/analyses
  • Restriction digestion
  • Gene cloning
  • Amplification
  • DNA sequencing
  • DNA profiling
  • Molecular biotechnology
  • Phylogenetic studies
• Common sources of DNA:

  • Whole blood
  • Hair
  • Sperm
  • Bones
  • Nails
  • Tissues
  • Blood stains
  • Saliva
  • Buccal (cheek) swabs
  • Epithelial cells
  • Urine
  • Paper cards with collected samples
  • Bacterial cultures
  • Fungal cultures
  • Animal tissues
  • Plants
• General steps for Nucleic Acid Isolation:

  1. Tissue homogenization and cell lysis:

  • Mechanical methods: sonication, grinding
  • Chemicals for extraction
  • Buffer (e.g. Tris-HCl)
  • Salt (e.g. NaCl)
  • Cell lysis reagents (e.g. SDS)
• General steps for Nucleic Acid Isolation:

  1. Tissue homogenization and cell lysis:

  • Denaturants (e.g. guanidium - inactivates RNases)
  • Enzymatic treatment (e.g. lysozyme, cellulase, pectinase)
• General steps for Nucleic Acid Isolation:

  2. Denaturation and separation of other biomolecules from the nucleic acid:

  • Chemical treatment
  • Phenol denatures proteins
  • Chloroform removes proteins and lipids
  • Isoamyl alcohol removes phenol and chloroform
  • CTAB (Cetyltrimethylammonium bromide) removes polysaccharides
  • PVP (polyvinylpyrrolidone) removes polyphenols
  • Enzymatic treatment (e.g. protease)
  • Centrifugation
• General steps for Nucleic Acid Isolation:

  3. Precipitation of nucleic acid from the aqueous phase:

  • Monovalent cations: sodium, potassium, ammonium
  • Alcohol: ethanol (95% to absolute), isopropanol
  • Centrifugation
• General steps for Nucleic Acid Isolation:

  4. Washing of precipitated nucleic acid:

  • 70% alcohol
  • Centrifugation
  • May be done two to three times
• General steps for Nucleic Acid Isolation:

  5. Drying of pellet and dissolution of dried pellet:

  • Air drying or vacuum drying
  • Dissolution in sterilized molecular grade water or TE (Tris-EDTA)
  • EDTA inactivates DNases
• After Isolation:

  • RNase treatment if DNA is isolated
  • DNase treatment if RNA is isolated
  • Storage:
  • Stock solution at -20˚C
  • Working solution at 4˚C
  • Determination of purity and concentration:
  • Using DNA standards (different concentrations)
  • UV spectrophotometry
  • Gel electrophoresis
• Purity determination:

  • Uses a UV Spectrophotometer
  • Determine A260 and A280
  • Compute for the ratio A260/A280
  • A260/A280:
  • 1.8 - 2.0: high purity of nucleic acid isolate
  • <1.8 (<1.6): protein contamination
  • >2.0: chloroform/phenol contamination
  • Accurate quantification not possible for crude samples
• Purity determination:

  • Another ratio used: A260/A230
  • A260/A230:
  • 2.0-2.2 or > A260/A280: pure isolate
  • <2.0: contaminated with carbohydrate carryover (especially in plants), residual phenol/guanidinium, other organic compounds, salts
• Quantifying Nucleic Acids:

  • Use the following formula:
  • [ds DNA] μg/mL = A260 × 50 × DF
  • [ss DNA] μg/mL = A260 × 37 × DF
  • [RNA] μg/mL = A260 × 40 × DF
• Gel Electrophoresis:

  • Movement of electrically charged molecules in an electric field
  • Separates molecules based on net charge and molecular weight
  • Commonly used gel: agarose
  • Visualizing agent: ethidium bromide, GelRed TM
• Staining with Ethidium Bromide:

  • Ethidium bromide is an intercalating agent
  • UV absorbed by DNA at 260 nm is transmitted to the dye
  • EtBr in UV light emits a red-orange color (fluorescence) at 590 nm detected by the naked eye
• Visualizing EtBr-stained Gels:

  • UV Transilluminator and Gel Documentation System