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• Nucleic acid extraction is the starting point in various downstream applications such as PCR, sequencing, DNA fingerprinting, and DNA microarray
• Nucleic acid extraction


  A series of steps to obtain nucleic acid samples (DNA/RNA) that are free from impurities and suitable for downstream application steps
• Purpose of nucleic acid extraction

  • Disintegrate the cell membrane
  • Active maximum elimination of lipids & proteins
  • Obtain pure DNA and/or RNA
  • Achieve high yield of DNA and/or RNA
• DNA Isolation


  Aims to get as much of the target out of the sample as possible
• Purification

  Reduces or eliminates contamination of the isolated target
• DNA Extraction

  One specific way to achieve isolation and purification using a solvent as an extractant, divided into different stages
• The aim of isolation and extraction is to recover nucleic acids
• Specific applications of DNA isolation/extraction

  • Scientific
  • Medicine
  • Forensic Science
• Different sources for DNA isolation

  • All types of living cells and fossilized cells
  • Semi-autonomous organelles containing DNA like mitochondria
  • Almost any intact cellular tissue including human & animal cells, plant cells, bacterial cells
• Basic steps in DNA isolation/extraction methods

  1. Cell and tissue disruption

  2. Removal of membrane lipids/lysis of cell membrane

  3. Protein denaturation and removal/digestion

  4. Removal of other cellular components

  5. RNA denaturation and removal

  6. DNA elution and storage (solid-phase extraction)
• Steps in DNA isolation/extraction

  1. Cell lysis

  2. Removal of contaminants like proteins, RNAs, other macromolecules

  3. Concentration of purified DNA/Purification of DNA

  4. Removal of membrane lipids

  5. Precipitation of DNA
• Cell and tissue disruption

  A method to break down the outer boundaries to release intercellular materials like DNA, RNA, proteins, organelles
• Cell lysis

  Mechanical methods include grinding, shearing, bead beating; Non-mechanical methods include physical (thermal lysis, sonification) and chemical (alkali [NaOH], detergents [SDS, CTAB], chaotropic agents [EDTA])
• Methods of cell lysis

  • Mechanical (grinding, shearing, bead beating)
  • Non-mechanical
  • Physical (thermal lysis, sonification)
  • Chemical (alkali [NaOH], detergents [SDS, CTAB], chaotropic agents [EDTA])
  • Enzymatic/Biological (proteinase K, lysozyme, lipase)
• After cell lysis

  The components of the cell are released
• Removal of Membrane Lipids

  1. By adding detergents

  2. Soaps and detergents penetrate the cell

  3. Soap lipids form small lipid droplets with the membrane molecules

  4. Membrane lipids are removed from the cell

  5. Removed from the sample when DNA is washed
• Protein Denaturation & Removal

  1. Protease for protein denaturation

  2. Soluble components of the proteins washed out in subsequent washings of the DNA

  3. Optional step

  4. Done by frequent washing
• RNA Denaturation & Removal

  1. RNA is an important contaminant of the DNA

  2. RNA is denatured using RNase
• Precipitation of DNA

  1. Separates the freed DNA from the cellular debris

  2. Adding high concentrations of salt (ammonium acetate) to DNA-containing solutions

  3. Salts neutralize the negative charges of the DNA molecule, making it more stable but less water-soluble

  4. Cations from salts counteract repulsion caused by the negative charge of the phosphate backbone

  5. DNA-salt mixture + Solvents (Ethanol or Isopropyl Alc) = DNA precipitation

  6. Solvents (ethanol or isopropanol) facilitate DNA precipitation

  7. DNA clumps together and precipitates out as a whitish gel

  8. Other salt contents remain in the aqueous solution
• Elution of DNA (silica-based method)

  Utilizes slightly alkaline buffer or double-distilled water
• Resuspension of DNA

  After DNA purification, DNA is resuspended in TE buffer (Tris-EDTA) or nuclease-free water (sterile)
• Advantages of TE buffer

  • Used for long-term DNA storage
  • Prevents DNA from being damaged by nucleases, inadequate pH, heavy metals, and oxidation by free radicals
  • Provides a safe pH of 7-8 (Tris)
  • EDTA chelates divalent ions used in nuclease activity & counteracts oxidative damage from heavy metals
  • Divalent ions: magnesium (serve as a cofactor for enzymes)
• Basic Criteria in Choosing DNA Isolation Methods

  • Efficient extraction
  • Sufficient amount of DNA extracted for downstream processes
  • Removal of contaminants
  • Quality and purity of DNA
• In alkali lysis, hydroxyl ions are used in lysing the cell membrane; break the fatty acid glycerol ester bonds in the cell membrane, making the cell membrane permeable
• Chaotropic agents (EDTA) can break the structure of DNA, thereby weakening the hydrophobic interactions
• Using oxyl ions in lysing the cell membrane

  Break the fatty acid glycerol ester bonds in the cell membrane, making it permeable
• Uses of oxyl ions in lysing the cell membrane

  • Isolating plasmid DNA from bacteria (E. coli)
• Chaotropic agents (EDTA) breaking the structure of DNA

  Weakening the hydrophobic interactions
• Advantage of enzymatic cell lysis

  • Specificity
• Lysozyme for bacterial cell lysis

  React with the peptidoglycan layer
• Method to break the cell wall of plants

  Combination method used (ex. Grinding + CTAB)
• CTAB

  • Most suitable for plant DNA extraction
• CTAB

  Cetyl Trimethyl Ammonium Bromide
• Compounds used for successful DNA Isolation

  • Detergents
• Proteinase K

  Digests proteins
• Chelating agents

  Bind to divalent cation and inactive DNase
• RNAse A

  Removes RNA contaminants
• Organic solvents

  To purify DNA in most common procedures
• Organic solvents used for DNA purification

  • Phenol
  • Chloroform
  • Isoamyl alcohol
• Phenol and chloroform

  Commonly used to separate proteins from DNA