BIO 101 - Module 3

• Why isolate nucleic acids?

  • High quality DNA is obtained for other 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 sample

  • bacterial cultures • fungal cultures

  • animal tissues • plants
• Steps in Nucleic Acid Isolation:

  1. Tissue Homogenization and Cell Lysis
  2. Denaturation and separation of other biomolecules from the nucleic acid
  3. Precipitation of nucleic acid from the aqueous phase
  4. Washing of the precipitated nucleic acid
  5. Drying of pellet and dissolution of dried pellet
• Tissue Homogenization and Cell Lysis

  • Mechanical method: sonication and grinding

  chemicals for extraction:

  • buffer (e.g. Tris-HCl)

  • salt (e.g. NaCl)

  • cell lysis reagents (e.g. SDS)
• • denaturants (e.g. guanidinium - inactivates RNases)

  • enzymatic treatment (e.g. lysozyme, cellulase, pectinase)
• 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
• 3. Precipitation of nucleic acid from the aqueous phase

  monovalent cations

  • sodium, potassium, ammonium

  alcohol

  • ethanol (95% to absolute)

  • isopropanol

  • centrifugation
• 4. Washing of precipitated nucleic acid

  • 70% alcohol

  • centrifugation

  • may be done for two to three times
• 5. Drying of pellet and dissolution of dried pellet

  • air drying or vacuum drying

  • dissolution in sterilized molecular grade water or TE (Tris-EDTA)

  • Ethylenediaminetetraacetic acid (EDTA) – inactivates DNases. How?
• After Isolation: Purification and Analysis

  • RNase treatment if DNA is isolated

  • DNase treatment if RNA is isolated

  Storage:

  • stock solution at -20C

  • working solution at 4C

  Determination of purity and concentration

  • using DNA standards (different concentrations)

  • UV spectrophotometry

  • Gel electrophoresis
• • Uses a UV Spectrophotometer

  • Determine A260 and A280

  • 260nm – λmax for nucleic acids

  • 280nm – λmax for proteins

  • Compute for the ratio A260/A280
• • A260/A280

  • 1.8 to 2.0 – high purity nucleic acid isolate

  • < 1.8 (< 1.6) – protein contamination

  • > 2.0 – chloroform contamination

  • Accurate quantification is not possible for crude samples
• Another ratio used: A260/A230

  • 230 nm – λmax for carbohydrates

  • 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
• [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 on the basis of net charge and molecular weight

  For nucleic acids:

  • Commonly used gel : agarose

  • Visualizing agent : ethidium bromide, GelRedTM
• Gel Electrophoresis

  • DNA migrates towards the anode

  • DNA is negatively charged

  “Blue Juice”

  • Contains buffers and dyes (bromophenol blue dye and Xylene cyanol)
• Gel Staining

  • Ethidium bromide

  -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 590nm detected by the naked eye
• Visualizing Stained Gels

  • UV Transilluminator and Gel Documentation System