capillary electrophoresis and the human genome
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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. - PowerPoint PPT PresentationTRANSCRIPT
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• 1990: DNA and RNA sequencing used
Sanger Method:1. Amplification – using E. coli2. Labeling – using radioactive ddntps3. Separation – using electrophoresis4. Reading – done manually
60 years would be needed to sequence the 3 billion base-pairs in human DNA
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
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
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
Speed verses Accuracy • The gel below exhibits “joule heating”,
distortions caused when the voltage applied is too high for the conditions used
Photo: Katie Kollitz 2008
Protein products from E. coli
Sample 96 Lane Gel Electrophoresis
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
Capillary Electrophoresis
http://en.wikipedia.org/wiki/File:Capillaryelectrophoresis.gif
Inside the Capillary• 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
Na+Na+ Na+ Na+ Na+
Na+Na+Na+Na+
Na+Na+Na+Na+
Na+Na+Na+Na+ Na+
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
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
Raw Results
Capillary gel electrophoresis of a DNA sequence using fluorescently tagged primer & ddCTP: spikes in voltage indiate the presence of a Cytosine residue
Four Color Fluoresence• Four color fluorescence allowed for data to
be read by a machine instead of manually
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
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
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