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 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. http://patients.about.com/od/yourdiagnosis/a/persmedicine.htm

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

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

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 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

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

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 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 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+ EM is a property of each analyte affected by charge and frictional drag EOF – Bulk Flow -generally larger than EM http://content.answers.com/main/content/img/McGrawHill/Encyclopedia/images/CE226400FG0010.gif

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 Swerdlow, H. and Gesteland, R. “Capillary gel electrophoresis for rapid, high resolution DNA sequencing.” Nucleic Acids Research. 18 (1990): 1415-1419. 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