DNA Isolation Techniques

DNA Isolation

• DNA isolation is a common procedure used in various fields including molecular biology and forensics
• First DNA isolation was conducted by Friedrich Miescher in 1869
• DNA extraction is used to purify DNA from samples by using physical and chemical methods

Steps involved in DNA Extraction

• Breaking cells open (Cell Lysis/Disruption):

  • This step exposes the DNA within the cells
  • Achieved through methods like detergent addition and physical disruption, which remove lipids and facilitate cell lysis
  • Removing protein with a protease (optional)
  • Removing RNA with RNase (almost always done)
  • This step also cleanses DNA from detergents, proteins, salts, and other reagents used during cell lysis

• DNA Purification:

  • Most commonly used DNA purification methods include:
    • Ethanol Precipitation: DNA is insoluble in ethanol and aggregates when exposed to it, forming a pellet after centrifugation
    • Phenol-Chloroform Extraction: Phenol denatures proteins and chloroform removes phenol residues, isolating DNA in the aqueous phase
    • Minicolumn Purification: Nucleic acids bind selectively to a silica-based solid phase under specific conditions, allowing for purification.

Refining DNA Extraction Techniques

• Using a chelating agent to bind divalent cations (Mg²⁺ and Ca²⁺) prevents DNA degradation by enzymes like DNase
• Removing cellular and histone proteins bound to DNA can be done via protease addition, precipitation with sodium/ammonium acetate, or extraction with a phenol-chloroform mixture
• After isolation, DNA is dissolved in a slightly alkaline buffer (e.g., TE buffer) or in ultra-pure water

Specific DNA Extraction Challenges

• Archaeological Samples: DNA is often partially degraded and requires specialized techniques
• Samples with PCR Inhibitors: Inhibitors like humic acid, indigo dyes, and hemoglobin require specific extraction methods to ensure successful PCR analysis
• Microorganisms with Thick Cell Walls: Isolating DNA from organisms like yeast requires techniques that can break through the cell wall

Isolating Extrachromosomal DNA

• Extrachromosomal DNA, such as plasmids, can be easily isolated by:

  • Lysis of the cells followed by precipitation of proteins
  • Chromosomal DNA is trapped in the insoluble fraction, leaving the plasmid DNA in the soluble fraction after centrifugation

• A Hirt DNA Extraction isolates all extrachromosomal DNA in mammalian cells:

  • This method removes high molecular weight nuclear DNA, leaving only low molecular weight mitochondrial DNA and potential viral episomes

Detecting and Quantifying DNA

• Diphenylamine (DPA) Test:

  • This test uses chemical hydrolysis of DNA to produce a blue-colored compound, confirming the presence of DNA.
  • It relies on the reaction of ω-hydroxylevulinyl aldehyde, a product of 2-deoxyribose decomposition, with diphenylamine

• Spectrophotometry:

  • Measuring DNA absorbance at 260 nm and 280 nm provides information about its purity and concentration.
  • A 260/280 ratio of 1.8 indicates relatively pure DNA, whereas a lower value suggests protein contamination.

• Agarose Gel Electrophoresis:

  • DNA is cut with restriction enzymes and run on a gel stained with ethidium bromide or another dye.
  • Comparing the intensity of the DNA bands to a DNA marker of known concentration allows for quantification.

• Southern Blot Technique:

  • This technique allows for further analysis of quantified DNA using PCR and RFLP techniques.
  • It helps identify and differentiate repeated sequences within the genome, making it valuable for forensic analysis.

Proteinase K

• An enzyme that digests proteins
• Isolated from fungi
• Active over a wide pH and temperature range, with increased activity at elevated temperatures (up to 55° C)
• Suited for short digestion times
• EDTA does not interfere with its action
• Ensures complete protein digestion
• Prevents degradation of DNA and RNA during purification by inactivating nucleases

2-Mercaptoethanol (β-mercaptoethanol, BME, etc.)

• A compound with the formula HOCH2CH2SH
• Used to reduce disulfide bonds and act as a biological antioxidant
• In RNA isolation, it denatures ribonucleases by reducing their disulfide bonds, preventing them from digesting RNA during extraction

Key Terms:

• 2' deoxyribonucleoside triphosphate: The building block of DNA, consisting of a deoxyribose sugar ring with three phosphate groups and a nitrogenous base.
• Daughter Strand: The newly synthesized strand of DNA during replication.
• DNA Helicase: Separates DNA strands during replication.
• DNA Ligase: Seals breaks or nicks in DNA strands.
• DNA Polymerase: Catalyzes the addition of nucleotides to DNA during replication.
• Primase: Initiates RNA primer synthesis on the lagging strand during replication.
• Holoenzyme: A complex of enzymes that work together, such as in DNA replication.
• Lagging Strand: The DNA strand synthesized discontinuously during replication.