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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
•
1990: DNA and RNA sequencing used
Sanger Method:
1.
2.
3.
4.
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
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
Protein products from E. coli
Photo: Katie Kollitz 2008
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
Na+
Na+
Na+
Na+
Na+
Na+
Na+
Na+
Na+
Na+
Na+
Na+
Na+
Na+
Na+
Na+
Na+
Na+
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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 Sequence
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 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