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
predisposition to a variety of illnesses, including breast cancer, disorders of hemostasis, cystic fibrosis, liver diseases and many others. Also, the etiologies for cancers, Alzheimer's disease and other areas of clinical interest are considered likely to benefit from genome information and possibly may lead in the long term to significant advances in their management.

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
Give more detail here

4. Sanger Method Advantages: Disadvantages:
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
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

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
Gel electrophoresis uses a porous semisolid matrix with aqueous buffer to separate large molecules by size alone
The pores act as a sieve through which smaller molecules migrate more quickly while 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
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
Capillary Electrophoresis applies a very high voltage to a narrow bore capillary (how narrow?) to separate molecules and ions
Advantages of CE:
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

11. 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+
EM is a property of each analyte affected by charge and frictional drag
EOF – Bulk Flow
-generally larger than EM

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

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