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Capillary Electrophoresis and the Human Genome

Capillary Electrophoresis and the Human Genome. Sequencing the Human Genome. Medical uses Genetic predisposition to diseases Forensic uses Matching DNA samples Potential for “personalized medicine” Testing for genetic abnormalities and customizing treatment. The Sequencing Challenge.

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Capillary Electrophoresis and the Human Genome

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  1. Capillary Electrophoresis and the Human Genome

  2. Sequencing the Human Genome • Medical uses • Genetic predisposition to diseases • Forensic uses • Matching DNA samples • Potential for “personalized medicine” • Testing for genetic abnormalities and customizing treatment

  3. The Sequencing Challenge • 1990: DNA and RNA sequencing used Sanger Method: • Amplification – using E. coli • Labeling – using radioactive ddntps • Separation – using electrophoresis • Reading – done manually 60 years would be needed to sequence the 3 billion base-pairs in human DNA

  4. Sanger Method • Advantages: • Automation to a certain degree • Disadvantages: • Time: 60 years would be needed to sequence the 3 billion base-pairs in human DNA

  5. Electrophoresis • Applied direct current to separate molecules based on charge and size • The higher the voltage, the faster separation will be achieved • Can be done in liquid or gel medium, slab or capillary

  6. Voltage and Speed of Separation • The migration rate v of an ion in cm/s is given by: v = μE • A higher electric field (E), achieved through a higher applied voltage, will result in a faster separation • μ can be changed only slightly using different conditions like pH, surfactants and buffers

  7. Speed verses Accuracy • The gel below exhibits “joule heating”, distortions caused when the voltage applied is too high for the conditions used Protein products from E. coli Photo: Katie Kollitz 2008

  8. Sample 96 Lane Gel Electrophoresis

  9. Capillary Electrophoresis • The surface area of a capillary is very high compared to its volume, so joule heating disappeared • The narrow capillary has a high resistivity, meaning current will stay low even at high voltages • Voltages are much higher in CE

  10. Capillary Electrophoresis http://en.wikipedia.org/wiki/File:Capillaryelectrophoresis.gif

  11. Inside the Capillary Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Electric Field provides separative power, causes bulk flow Electrophoretic Mobility: property of each analyte Electroosmotic Flow: allows for elution and separaton of positive, neutral and negative molecules http://content.answers.com/main/content/img/McGrawHill/Encyclopedia/images/CE226400FG0010.gif

  12. 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

  13. Capillary Gel Electrophoresis • Since DNA has a uniform charge to size ratio, a gel must be used to introduce frictional forces • CGE retains the advantages of speed, small sample size and quantitative output

  14. Raw Results Capillary gel electrophoresis of a DNA sequence using fluorescently tagged primer & ddCTP: spikes in voltage indiate the presence of a Cytosine residue

  15. Four Color Fluoresence • Four color fluorescence allowed for data to be read by a machine instead of manually

  16. Future Developments • Concept of lab on a chip • Preparation step is the only one that hasn’t been automated • Lab on a chip eliminates amplification step and separation step • Labeling and reading happen simultaneously • Requires intense computational ability

  17. True Signal Molecule Sequenceby 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 and position of ddNTs recorded. • Remove fleorescently tagged group leaving behind generated complementary strands • Repeat for other bases

  18. Results • Multiple four base cycles provide 25 base length sequences! Fluoresecence of 1 and 2 in cycle X indicate the presence of a G nucleotide

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