CE & Genome

Capillary Gel Electrophoresis and the Human Genome

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Uses of Human Genome Sequence

Medical uses: genetic predisposition to diseases
Forensic uses: matching DNA samples
Potential for “personalized medicine”: Testing for genetic abnormalities & adjusting therapies

Classic Sequencing: Sanger Method

Steps of Sanger Method

Amplification – using E. coli
Labeling – using radioactive ddntps
Separation – using electrophoresis
Reading – done manually

Advantages

Automation to a certain degree

Disadvantages

Time: 60 years would be needed to sequence the
3 billion base-pairs in human DNA

Electrophoresis

Apply 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

The migration rate (v) of an ion in cm/s is given by:

v = μE

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

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

Gel Electrophoresis

Uses a porous semisolid matrix with aqueous buffer to separate large molecules by size alone

Smaller molecules migrate more quickly through pores
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

joule heating: distortions caused when the voltage applied is too high for the conditions used

The electric field must be kept low to avoid joule heating, or uneven heating in the gel that causes distortions

Gel distorted by joule heating resulting in uneven bands
and width of columns

Capillary Electrophoresis

Diagram of Typical Capillary Electrophoresis. Analytes migrate
through capillary from source vial to destination vial due to applied electric field.

The narrow capillary has a high resistivity, meaning current will stay low even at high voltages
Voltages are much higher in CE

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
No joule heating! The surface area of a capillary is very high compared to its volume.

Inside a capillary

Electric Field provides separative power, causes bulk flow

Electrophoretic Mobility: a property of each analyte affected by charge and frictional drag

Electroosmotic Flow: allows for elution and separaton of positive, neutral and negative molecules

Diagram of inside a capillary tube. Analytes travel form anode to
cathode and interact with inside of tube to varyign degrees. Negatively
charge analytes elute first, followed by nonpolar and positively charged.

Capillary Gel Electrophoresis

Since DNA has a uniform charge
to size ratio, a gel must be used to introduce frictional forces

Advantages:

speed
small sample size
quantitative output

Capillary gel electrophoresis of a DNA sequence using fluorescently tagged primer & ddCTP: spikes in voltage indiate the presence of a Cytosine residue (Swerdlow and Gesteland 1990)

Four Color Fluorescence

Four color fluorescence allowed for data to be read by a machine instead of manually

Future Developments: True Single Molecule Signaling 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

Image take and position of ddNTs recorded.

Remove flourescently tagged group cleaved off

Repeat for other fluorescently tagged NTs

Multiple four base cycles provide 25 base length sequences!

Fluoresecence of 1 and 2 in cycle X indicate the presence of a G nucleotide.

References

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

http://www.helicosbio.com/Technology/TrueSingleMoleculeSequencing/tabid/64/Default.aspx