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Analyzing DNA with Capillary Electrophoresis.

Capillary electrophoresis offers several advantages over traditional slab-gel electrophoresis in analyzing DNA molecules and components. It is faster, less labor-intensive, readily automatable, and can require only picogram samples rather than the microgram quantities needed by slab systems.

Generally speaking, DNA segments less than 10 bases in length can be analyzed using either capillary zone electrophoresis (CZE) or micellar electrokinetic chromatography (MEKC). Separations of larger molecules require chemical or physical gels.

Capillary zone electrophoresis separates DNA molecules based on the charge-to-mass ratios of the analytes, and its selectivity is governed by differences in each analyte's effective mobility.

CZE uses fused-silica capillaries that typically have internal diameters of 20 to 100 mm and lengths of 20 to 100 cm to achieve the high surface-to-volume ratios needed for efficient dissipation of Joule heating generated by the flow of electrical currents in response to high electric fields.

For a CZE separation, a capillary is filled with an appropriate buffer at the desired pH. Both ends of the capillary are then placed into buffer reservoirs, and up to 30,000 V is applied between them. When the sample is introduced, the ionic species in the sample plug migrate with an electrophoretic mobility that's determined by their charge and mass. If the applied field were the only force acting on the molecules, ions of one electrical sign would start moving toward the detector, while ions of the opposite sign moved away and neutral molecules remained stationary, and thus CZE would not be a very useful technique. In actuality, the buffer solution itself begins moving toward the DNA detector in response to an electroosmotic force, thereby transporting all of the separated analytes in the detector's direction.

This electroosmotic "force" is the result of a complex set of circumstances. Ionized silanol groups at the capillary wall attract cationic species from the buffer, an effect that is regulated by the pH of the buffer. The ionic layer that is formed has a positive charge density that decreases exponentially with distance from the wall. The portion of the ionic layer closest to the wall remains essentially stationary while cations in the other portion migrate in response to the applied field, carrying waters of hydration with them. Because of the cohesive nature of the hydrogen bonding of the waters of hydration to the water molecules of the bulk solution, the entire buffer solution is pulled toward the cathode.

Micellar electrokinetic chromatography was originally developed to separate neutral compounds, but now is used increasingly to separate charged compounds that have similar electrophoretic mobilities. In this approach, micelles are added to the buffer to interact with the analytes ways that will change their mobilities. Micelles are molecular aggregates of surfactant molecules that partition the analytes according to hydrophobicity, ionic attraction, and hydrogen bonding.

Generally, neutral pH is used when analyzing nucleic acids. Under this condition, the bases and nucleosides are uncharged and their separation results from differential partitioning within the micelles.

With increasing size, DNA molecules begin to have a mobility that's independent of molecular weight, because of their constant linear charge density. This means that a sieving medium such as a gel must be used to separate the molecules according to their size. The gel works by impeding the travel of large molecules more than that of small ones.

Chemical gels consist of sieving matrixes that are cross-linked and/or chemically linked to the capillary wall. This chemical linking takes place after the matrix has been flowed into the capillary, so the media becomes stuck inside the capillary. When the media is no longer functional, neither is the capillary. The resolving power of the combination depends on the field strength and length of the capillary, with longer capillaries giving higher resolution at the expense of separation time.

While chemical gels have proven to be highly effective in DNA separations, there have been enough problems with the technique to motivate the search for replaceable physical gels.

Physical gels are solutions of hydrophilic polymers dissolved in an appropriate buffer. The polymers that are used are not cross-linked, so they can be pumped out of the capillary at the end of each run, allowing a fresh separation medium to be used for each analysis. The individual strands of a physical gel interact with their neighbors, thereby creating a mesh possessing a sieving capability. In addition to entangling the DNA molecules, it's possible for the mesh to undergo specific interactions with DNA that are sensitive to polymer length and structure.
More Info

Amersham Pharmacia Biotect
Harry's CE Page         
Janis Research Co.      
Capillary Electrophoresis
  Discussion Group
Separation Science Group,
  Univ. of Tasmania (Australia)
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Author:Comello, Vic
Publication:R & D
Article Type:Brief Article
Date:Jan 1, 2000
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