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. coli

2. Labeling – using radioactive ddntps

3. Separation – using electrophoresis

4. 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