3.3 DNA isolation and purification techniques

DNA isolation and purification are crucial steps in biotechnology. These techniques allow scientists to extract genetic material from cells, separating it from other cellular components. By mastering these methods, researchers can obtain pure DNA samples for further analysis and manipulation.

From cell lysis to advanced purification techniques, this section covers the essential processes for obtaining high-quality DNA. Understanding these methods is key to successful genetic research and applications in biotechnology, forming the foundation for many downstream experiments and analyses.

Cell Lysis and Extraction

Cell Lysis Methods

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• Cell lysis involves disrupting the cell membrane to release the cell contents, including DNA
• Mechanical methods of cell lysis include sonication (using high-frequency sound waves), homogenization (using a blender or pestle), and freeze-thaw cycles (alternating between freezing and thawing)
• Chemical methods of cell lysis use detergents (SDS, Triton X-100) or enzymes (lysozyme, proteinase K) to break down the cell membrane and release the DNA
• Osmotic lysis uses a hypotonic solution to cause the cells to swell and burst due to the influx of water

Phenol-Chloroform Extraction and Ethanol Precipitation

• Phenol-chloroform extraction separates DNA from proteins and other cellular components
  • Phenol denatures proteins and separates them from the aqueous phase containing DNA
  • Chloroform helps stabilize the phenol and facilitates the separation of the organic and aqueous phases
• Ethanol precipitation concentrates and purifies the DNA
  • Addition of salt (sodium acetate) and cold ethanol causes the DNA to precipitate out of solution
  • The precipitated DNA can be collected by centrifugation and resuspended in an appropriate buffer
• Centrifugation is used throughout the extraction process to separate the phases and pellet the DNA
  • High-speed centrifugation (10,000-15,000 rpm) is required to effectively pellet the DNA

Purification Techniques

Column Chromatography

• Column chromatography separates molecules based on their interaction with a stationary phase (column material) and a mobile phase (buffer)
• Affinity chromatography uses a ligand (antibody, enzyme, or receptor) immobilized on the column to specifically bind the target DNA
  • The bound DNA can be eluted by changing the buffer conditions (pH, salt concentration)
• Ion-exchange chromatography separates DNA based on its charge
  • Positively charged columns (anion-exchange) bind negatively charged DNA
  • Negatively charged columns (cation-exchange) bind positively charged molecules
• Size-exclusion chromatography separates DNA based on its size
  • Smaller molecules are retained in the pores of the column material, while larger molecules pass through quickly

Gel Electrophoresis

• Gel electrophoresis separates DNA fragments based on their size and charge
• DNA is negatively charged due to the phosphate backbone and migrates towards the positive electrode (anode)
• Agarose or polyacrylamide gels act as a molecular sieve, with smaller fragments moving faster through the gel matrix
• The DNA fragments are visualized by staining with ethidium bromide or other DNA-binding dyes and exposure to UV light
• The size of the fragments can be determined by comparing their migration to a DNA ladder (a set of fragments of known sizes)

Quantification

Spectrophotometry

• Spectrophotometry measures the absorption of light by a sample at a specific wavelength
• DNA absorbs UV light at 260 nm, while proteins absorb at 280 nm
• The ratio of A260/A280 provides an estimate of the purity of the DNA sample
  • A ratio of ~1.8 indicates pure DNA, while lower values suggest protein contamination
• The concentration of DNA can be calculated using the Beer-Lambert law: A = εbc
  • A is the absorbance, ε is the extinction coefficient (50 ng-cm/μL for double-stranded DNA), b is the path length (usually 1 cm), and c is the concentration
• Spectrophotometry is a quick and simple method for quantifying DNA but may overestimate the concentration if RNA or other contaminants are present