Capillary Electrophoresis

Capillary electrophoresis (CE) is an extremely powerful instrumental technique that has introduced automation into the clinical molecular laboratory. The method, originally described by Jorgenson and colleagues at the University of North Carolina in 1983 (7), utilizes electrophoresis in the thin, fused silica capillary columns that had been recently developed for use in capillary gas chromatography. Electrophoresis in free solution (without porous matrices) in open tubes was not new, having been described by Tiselius in 1937 (8). In the original format, however, resolution was severely compromised by the heat produced by the passage of the electric current though the solution, as the uneven heating created unwanted mixing within the electrophoresis tube. The introduction of the capillary tube solved the Joule heating problem, as the capillaries used (with inner diameters of 50-100 |im) have a very high surface-to-volume ratio and are extremely efficient at dissipating heat, even at applied voltages of 10,000-20,000 V.

Detection of the separated products is accomplished with detectors similar to those used in high-performance liquid chro-motography (HPLC). Because the detector is fixed in position near the end of the capillary column, an analyte must pass through the entire length of the capillary before being detected. This represents a significant advantage in terms of resolution compared to slab gels. In CE, each peak is detected only after having experienced the separating power of the entire column. In contrast, in slab gels, bands obviously must remain on the gel to be detected, with the more difficult to resolve, higher-molecular weight species being the ones that have migrated the least distance into the gel, thereby experiencing the least amount of separation. Because the fused silica capillaries are optically clear (after removal of a small window in the poly-imide coating that capillaries are supplied with in order to prevent breakage), detection can take place directly in the column; there is not need for any connective plumbing between the column and the detector. This has significant advantages in terms of both instrumental simplicity and elimination of any band-broadening effects as a result of an imperfect fluid path connections. Although ultraviolet (UV)-visible detectors can be used to monitor DNA separations, the majority of applications utilize laser-induced fluorescence (LIF) detectors. Because DNA has very low intrinsic fluorescence, the DNA must be labeled with a fluorescent tag, typically by amplifying the target with a labeled PCR primer, in order to use an LIF detector. Modern CE-LIF detectors can monitor four of more different emission wavelengths; thus, in addition to being an extremely sensitive detection method, fluorescence has the great advantage of being compatible with multiplexing, or analyzing several different PCR products at once. A generalized schematic of a CE instrument is shown in Fig. 3.

Although CE was rapidly shown to be a powerful separation tools for small molecules, the application to macromolecules, proteins, and nucleic acids required the solution to several fundamental problems. The surface of the silica column is compose of large numbers of silanol groups (-Si-OH). At high pH, these slightly acidic hydroxyls ionize, giving a surface with a high net negative charge. This negative charge attracts cations from the buffer. These cations are bound to the charged silica wall in two identifiable layers, the Inner Helmholtz or Stern Layer, and the Outer Helmholtz Plane (OHP). The inner layer is very tightly bound and is essentially static, but when an electric field is applied, the positive ions of the OHP, along with their water of hydration, migrate to the cathode. As the waters of hydration are hydrogen-bonded to the water molecules of the bulk solution inside the capillary, there is a net flow of liquid, or buffer, from the anode to the cathode. This bulk flow of buffer within the capillary column on application of an electric field is the endo-osmotic flow (EOF) (9). The EOF can be manipulated, typically be alterations in the buffer pH, and is a useful tool in designing separations of mixtures of small molecules that are heterogeneous in structure and charge. For nucleic acids, however, which are uniform in structure and charge, the EOF is highly detrimental to size-based separations. Another problem with separations of macromolecules by CE is the pronounced tendency for these molecules to interact with the column wall, sometimes irreversibly, but always with a drastic and detrimental effect on the peak shapes and separation efficiency. In addition, elec-trophoresis is typically used to separate compounds that differ from each other by charge or shape. In the case of nucleic acids, two of the terms in Eq. (2), the frictional coefficient and the charge, change together as a function of the size of the nucleic acid fragment (i.e., DNA molecules display a constant frictional coefficient-to-charge ratio). Thus, there is no separation of different sizes of DNA molecules by electrophoresis without a sieving matrix.

Fortunately, these three problems have been solved. The first two problems, elimination of the EOF and adsorption of macromolecules to the column wall, have the same solution— coating the interior wall of the column with a noncharged coating. There have been a variety of column-coating methods utilized, including those borrowed from the capillary gas chromatography field where capillary coatings, with a wide variety of polymers, are used as solid phases for chromato-graphic separations. Other methods include derivitization of the negatively charged silanol residues on the surface of the silica column with silating reagents.

Microsatellite Electrophoresis

Outlet Buffer Reservoir

Capillary Instability

Data Analysis and Storage

Fig. 3. A schematic of a capillary electrophoresis instrument. The basic components of a CE system are a high-voltage (5-30 kV) power supply, a polyimide-coated, fused-silica capillary column (20-100 |lm inner diameter, 20-50 in length) filled with buffer, buffer reservoirs to accommodate both the column and the electrodes, a detector (nucleic acid applications typically utilize a LIF detector), and a desktop computer for instrument programming and data analysis and storage.

Sample / Inlet Buffer Reservoir

The first attempts to introduce a sieving matrix into a capillary column were a variety of methods to polymerize acry-lamide inside the column. Although it is possible to prepare a gel-filled column, the technique was plagued with problems, including the formation of bubbles during polymerization, which block the electric current, extrusion of the gel during electrophoresis by the EOF, and limited number of times the column could be used. In 1993, Karger and colleagues at Boston University introduced the notion of replaceable sieving matrices. These matrices consisted of, not a gel of bis-acrylamide crosslinked polyacrylamide, but a viscous solution of non-crosslinked, high-molecular-weight linear polyacrylamide (10). At concentrations greater than the entanglement threshold, these polymer solutions acted in a very similar way to gels in DNA size separations. In gels, the size-based separation is mediated by the pore size. In solutions of water-soluble polymers, the separation is mediated by the pores between entangled polymer chains. This breakthrough enabled the development of commercial, capillary-based sequence analyzers using coated capillaries and linear acrylamide polymer solutions.

The systems using coated capillaries and linear polyacrylamide are successful at providing DNA sequencing reads of well over 500 bases and form the basis for several commercial CE-based automated sequence analyzers. However, the coated capillaries are relatively expensive to prepare and have a modest lifetime (approx 200 runs). More recently, a variety of polymers, including polydimethylacrylamide, polyethylene oxide, polyvinylpryolodone, and the recently described polyhydrox-yethylacrylamide (11-13) have been shown to not only provide sieving-based DNA size separations but also self-coating characteristics that allow them to be used with bare silica capillary columns. The details on the mechanisms of polymer coating of silica surfaces is still somewhat obscure, but apparently they involve both interactions between hydrophobic domains of the polymer and the hydrophobic siloxanes of the silica surface and hydrogen-bonding between the polymer and the surface silanols (14,15). The read length obtainable using a variety of polymers has been investigated and it is clear that linear poly-acrylamide is superior in terms of read length. The price, however, is paid in terms of the requirement to use the more expensive, shorter-lifespan coated columns. Currently, CE systems with DNA analysis capabilities are available from several manufacturers.

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