Fundamentals Of Electrophoresis

Electrophoresis refers to the migration of charged molecules through a liquid or gel medium under the influence of an electric field. Zone electrophoresis, or the migration of macromol-ecules through a porous support medium, or gel, is almost universally used in molecular biology laboratories today. Electrophoresis (all discussion in this chapter will involve zone electrophoresis but will be referred to as electrophoresis for brevity) is a powerful separation tool, being able to detect the differential migration of macromolecules with only subtly different structures. The two formats that are used are slab gel electrophoresis, in which the electrophoresis takes place in support medium, agarose, or polyacrylamide, and capillary electrophoresis (CE), an instrumental technique in which the electrophoresis takes place in a capillary tube.

The rate of migration of a macromolecule through a gel matrix is dependent on several factors, including (1) the net charge on the molecule at the pH at which the assay is conducted, (2) the size and shape of the molecule, (3) the electric field strength or voltage drop, (4) the pore size of the gel, and (5) temperature. The forces acting on the analyte to drive it through the gel are the charge on the molecule and the electric field strength. Equation

(1) describes the electrophoretic driving force (Table 1).

The forces acting to retard the movement of the molecule are the frictional forces, determined by the velocity of the ana-lyte, the pore size of the gel, and the size and shape of the molecule. The opposition of the acceleration of the analyte by the frictional forces is described by Stokes' Law as shown in Eq.

(2) (Table 1). When the electrophoretic driving force equals the frictional force (F = F), the result is a constant velocity of the analyte molecule through the gel matrix [Eq. (2), Table 1]. The term viX describes the velocity of the analyte through the gel matrix (cmis) per unit field strength (Vicm) under constant conditions (i.e., the same buffer conditions and the same gel viscosity). This term (given by the symbol ||) is defined as the electrophoretic mobility of the analyte (expressed as cm2iV-s). From the units of the electrophoretic mobility, it can be seen that if the gel is run under constant-voltage conditions (Vicm constant) for a given period of time (s), then (cm2iV-s)(V-sicm) is distance of migration (in cm). Thus, if gels are run such that the product of the voltage and the time are constant, the same analyte will migrate the same distance into the gel. For convenience, this is typically expressed as volt-hours. For example, if one gel is run at 50 V for 10 h (500 V-h) and an identical gel (constant length, viscosity, and buffer concentration) is run at 100 V for 5 h (500 V-h), the same analyte will appear at the same position on both gels. This ability to reproduce gel profiles is one of the principal reasons that constant-voltage run conditions are preferred for DNA analysis. An exception to this is the preferred conditions for DNA sequencing. As will be discussed later, sequencing gels are run at elevated temperature to ensure the adequate denaturation of the single-stranded DNA molecules; in this case, running the gel at constant watts is helpful in maintaining an even heating of the gel.

In protein analysis, the pH of the electrophoresis buffer can be a powerful tool in optimizing specific separations. This is because the type (acidic or basic) and number of ionizable groups found on proteins are variable. However, for DNA analysis, it is the charge of the phosphate backbone that is dominant. Therefore, DNA electrophoresis is typically performed at a slightly alkaline pH to ensure full ionization of the phosphate residues.

3.1. THE GEL MATRIX: AGAROSE There are two types of gel matrix in common use in DNA laboratories: agarose and polyacrylamide. Agarose is a polysaccharide commercially derived from seaweed. The agarose polymer consists of multiple agarbiose molecules linked together into linear chains, with an average molecular weight of 120,000 daltons. The agarbiose subunit is a disaccharide consisting of P-d-galactose and 3,6-anhydro-a-l-galactose (Fig. 1) (6). The partially purified material, agar, consists of noncharged polymer chains, agarose, and negatively charged chains. The negative charges are typically the result of sulfate (-SO4) residues. In general, the more highly purified the agarose, the lower the sulfate concentration, the higher the quality of the separation, and the higher the price. Agarose is supplied as a white, nonhydroscopic powder. A gel is prepared by mixing agarose powder with buffer, boiling the mixture, pouring the molten gel into a casting tray, and cooling. During this process, the agarose chains shift from existing in solution as random coils to a structure in which the chains are bundled into double helices. The average pore size for agarose gels are typically in the range 100-300 nm3. Agarose gels are used at concentrations near 1% (wiv) for separating DNA fragments in the 1- to 20-kb size range. Examples of applications common in the DNA diagnostic laboratory include restriction digestion analysis of large plasmids and Southern transfer analysis of genomic DNA.

Microsatellite Instability

Fig. 2. Schematic demonstrating the polymerization of acrylamide and bis-acrylamide monomers.

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