Capillary Gel Electrophoresis and the Human Genome

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Uses of Human Genome Sequence


Classic Sequencing: Sanger Method


Steps of Sanger Method
  1. Amplification – using E. coli
  2. Labeling – using radioactive ddntps
  3. Separation – using electrophoresis
  4. Reading – done manually
Advantages
  • Automation to a certain degree
Disadvantages
  • Time: 60 years would be needed to sequence the
  • 3 billion base-pairs in human DNA

Electrophoresis
 v = μE

where E is the electric field in v/cm and μ is the electrophoretic mobility of a molecule,
proportional to charge and inversely proportional to retarding factors like friction
Gel Electrophoresis
  • Uses a porous semisolid matrix with aqueous buffer to separate large molecules by size alone
    • Smaller molecules migrate more quickly through pores
    • Larger molecules will be impeded
  • Molecules must have a similar charge to size ratio
  • SDS can be used to give proteins a uniform negative charge to size, while DNA already has a uniformly negative charge to size ratio
  • In Gel electrophoresis, the electric field must be kept low to avoid joule heating, or uneven heating in the gel that causes distortions
    • joule heating: distortions caused when the voltage applied is too high for the conditions used
  • The electric field must be kept low to avoid joule heating, or uneven heating in the gel that causes distortions



Gel distorted by joule heating resulting in uneven bands
and width of columns




Capillary Electrophoresis



Diagram of Typical Capillary Electrophoresis.  Analytes migrate
through capillary from source vial to destination vial due to applied electric field.
  • The narrow capillary has a high resistivity, meaning current will stay low even at high voltages
  • Voltages are much higher in CE
Advantages
  • The small size of a capillary offers high resistance, so the electric field can be large while keeping current low, speeding separations and improving resolution
  • A smaller sample size may be used
  • Because the samples elute from one end, quantitative detectors may be used
  • Electroosmotic flow allows for separation of negative, positive and uncharged molecules
  • No joule heating! The surface area of a capillary is very high compared to its volume. 

Inside a capillary


  • Electric Field provides separative power, causes bulk flow
  • Electrophoretic Mobility: a property of each analyte affected by charge and frictional drag
  • Electroosmotic Flow: allows for elution and separaton of positive, neutral and negative molecules


Diagram of inside a capillary tube.  Analytes travel form anode to
cathode and interact with inside of tube to varyign degrees.  Negatively
charge analytes elute first, followed by nonpolar and positively charged. 

Capillary Gel Electrophoresis



Since DNA has a uniform charge
to size ratio, a gel must be used to introduce frictional forces

Advantages:
  • speed
  • small sample size
  • quantitative output



Capillary gel electrophoresis of a DNA sequence using fluorescently tagged primer & ddCTP: spikes in voltage indiate the presence of a Cytosine residue (Swerdlow and Gesteland 1990)





Four Color Fluorescence


Four color fluorescence allowed for data to be read by a machine instead of manually



Future Developments: True Single Molecule Signaling by Helicos BioSciences

  • Two flow cells filled with billions of copies of sample DNA attached to surface.
  • DNA polymerase catayzes reaction using one added fluorescently tagged ddNT.
  • Wash out free nucleotides
  • Image take and position of ddNTs recorded.
  • Remove flourescently tagged group cleaved off
  • Repeat for other fluorescently tagged NTs

  • Multiple four base cycles provide 25 base length sequences!

Fluoresecence of 1 and 2 in cycle X indicate the presence of a G nucleotide.


References

Swerdlow, H. and Gesteland, R.  Capillary gel electrophoresis for rapid, high resolution DNA sequencing.” Nucleic Acids Research.  18 (1990): 1415-1419. 


http://www.helicosbio.com/Technology/TrueSingleMoleculeSequencing/tabid/64/Default.aspx