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Molecular Biology

Electrophoresis

The two main workhorses for separating nucleic acids in the molecular lab: traditional Gel Electrophoresis (using agarose or polyacrylamide slabs) and the more modern Capillary Electrophoresis. Both use electric fields to move negatively charged DNA/RNA, but they differ significantly in format, resolution, speed, and automation

Think of it like comparing different ways to run a race:

  • Gel Electrophoresis: Like running an obstacle course on an open field (the gel slab)
  • Capillary Electrophoresis: Like running a high-speed race through a narrow, precisely engineered tunnel (the capillary)

Electrophoresis: The Core Concept

The fundamental principle for both is: * Negative Charge: DNA and RNA have a negatively charged phosphate backbone * Electric Field: When placed in an electric field, these molecules move towards the positive pole (anode) * Sieving Matrix: A medium (the gel or polymer solution) impedes their movement * Size Separation: Smaller molecules navigate the matrix faster and travel further than larger molecules in a given time

Gel Electrophoresis (The Slab)

This is the classic method, involving casting a slab of gel material and running samples through it in a buffer tank

  • Types & Characteristics
    • Agarose Gel
      • Matrix: Natural polysaccharide (from seaweed), easy to prepare
      • Format: Horizontal (“submarine”)
      • Resolution: Moderate; best for separating larger fragments (roughly 50 bp to >20 kb) that differ significantly in size. Pore size adjustable by % agarose
      • Use: Very common for checking PCR product size/presence, basic RFLP analysis
      • Detection: Post-run staining (e.g., Ethidium Bromide - mutagen, SYBR Safe - safer). Visualize under UV/blue light
    • Polyacrylamide Gel (PAGE)
      • Matrix: Synthetic polymer; more complex to prepare, monomer is toxic
      • Format: Vertical (thin slab between glass plates)
      • Resolution: High; excellent for separating small fragments (down to ~5 bp) and resolving single-base differences
      • Use: Historically vital for Sanger sequencing, mutation detection (SSCP), analysis of very small fragments
      • Detection: Staining (as above) or historically autoradiography
  • Workflow: Prepare gel -> Load samples (mixed with loading dye) & ladder -> Run in buffer tank with power supply -> Stain gel -> Visualize bands
  • Pros: Relatively inexpensive equipment, visually intuitive results, versatile for wide size ranges (especially agarose)
  • Cons: More hands-on time, slower run times, lower resolution (agarose), potential safety hazards (EtBr, acrylamide monomer), results less quantitative, not easily automated

Capillary Electrophoresis (CE) (The Tube)

This technique performs electrophoresis inside a very narrow (capillary) tube filled with buffer and a polymer solution, offering significant advantages in speed, resolution, and automation

  • Matrix & Format
    • Matrix: Typically a replaceable, pumpable polymer solution (e.g., linear polyacrylamide) inside a narrow fused-silica capillary. Acts as the sieving medium
    • Format: Automated instrument with single or multiple capillaries (arrays)
  • Separation: Based on size as fragments reptate (snake) through the polymer matrix under high voltage. Electroosmotic flow (EOF) is another factor influencing migration, often suppressed or controlled for DNA sizing
  • Resolution: Extremely high, often capable of resolving fragments differing by only one base pair
  • Use: The gold standard for:
    • Sanger Sequencing: Separating fluorescently labeled fragments to read DNA sequence
    • Fragment Analysis: Precisely sizing PCR products for STR analysis (forensics, chimerism), MLPA (copy number variation)
  • Detection: Laser-Induced Fluorescence (LIF). DNA fragments are fluorescently labeled (e.g., primers, ddNTPs). As they pass a detector window, a laser excites the dye, and emitted light is measured. Allows for multi-color detection (crucial for sequencing/multiplexing)
  • Workflow: Prepare samples (often PCR products with fluorescent labels) -> Load onto autosampler plate -> Instrument automatically injects sample into capillary -> High voltage separation -> On-column LIF detection -> Data output as an electropherogram (peaks vs. time)
  • Pros: Very high resolution, fast analysis time, fully automated (high throughput with arrays), highly sensitive (LIF), quantitative data (peak height/area), reduced reagent use
  • Cons: Very high instrument cost, requires specialized reagents, capillaries can clog or degrade, requires skilled operation/maintenance

Summary: Gel vs. Capillary

Feature Gel Electrophoresis (Slab) Capillary Electrophoresis (Tube)
Format Horizontal/Vertical Slab Narrow Capillary
Matrix Cast Agarose or Polyacrylamide Pumpable Polymer Solution
Resolution Moderate (Agarose) to High (PAGE) Very High (often 1 bp)
Speed Slower (Hours) Faster (Minutes)
Automation Low High (Fully automated)
Detection Post-run Staining (EtBr, SYBR) On-column Laser-Induced Fluorescence (LIF)
Primary Use PCR checks, RFLP, Prep work Sanger Sequencing, Fragment Analysis (STRs)
Cost Lower Instrument Cost High Instrument Cost
Output Image of Bands Electropherogram (Peaks)