Electrophoresis

Electrophoresis uses an electric field to separate charged molecules by differential mobility in a sieving matrix that can be either liquid or solid (gel). The differential mobility is determined by the size of the molecule and its conformation, the net charge of the molecule (as modified by pH), temperature, and the pore size of the matrix. DNA, being negatively charged, migrates towards the anode (+) when an electric field is applied to an electrolyte solution. The size of DNA can be modified by restriction endonuclease digestion (see below), rendering DNA fragments small enough to be mobile in the matrix. Conformation can be modified with denaturing conditions prior to or during electrophoresis. Nucleic acids are usually electrophoresed at a slightly alkaline pH to ionize all phosphate groups in the backbone of the molecule.

The pore size of the matrix is determined by the composition and concentration of the polymer. For any given pore size, the mobility of a molecule through the matrix is inversely proportional to the log of its size. Therefore, for a given size difference between two molecules, the difference in the rate of migration will be substantially less if both molecules are large. The limiting mobility is defined as the rate of migration through the gel at which large molecules can no longer be separated for any given pore size. This may be related to the tendency of sections of long DNA fragments to "snake" through different pores in the gel, retarding the mobility of the fragment. The limiting mobility of gels can be overcome by using pulsed field gel electrophoresis (PFGE), in which the voltage gradient is periodically reoriented.

Polyacrylamide Gels

In clinical molecular laboratories, the matrices used most commonly are acrylamide and agarose. Polyacrylamide gels are formed by cross-linking acrylamide monomers with bisacrylamide in the same salt buffer used for elec-trophoresis and pouring the solution in a thin space between two glass plates. A comb is inserted at one edge between the plates to form wells for sample insertion. After the gel has formed, the plates are mounted in a vertical electrophoresis unit such that the gel forms a bridge between two buffer chambers. Samples, controls, and sizing standards are loaded into the wells, usually in association with a dye to track the progress of electrophoresis, and glycerol to make the samples sink to the bottom of the wells. Electrodes are attached to the buffer chambers and connected to a power supply providing constant voltage. After electrophoresis, the glass plates are separated and the gel is soaked in EtBr solution. DNA is visualized by EtBr staining under UV light. Polyacrylamide forms very small pores and is useful for high resolution of DNA fragments from 100 to 1000 bp. Single base pair resolution can be achieved, allowing polyacrylamide gels to be used for sequencing under denaturing conditions (see below). However, polyacrylamide gels are thin and fragile, the glass plates are cumbersome to work with, and nonpolymerized acrylamide is a lung irritant and neurotoxin; therefore, alternatives to polyacrylamide gel electrophoresis are desirable in the clinical laboratory. Although more costly, precast acrylamide gels are commerically available to circumvent the biohazards of nonpolymerized acrylamide.

Agarose Gels

Agarose gels are formed by boiling an agarose gel powder until the agarose has completely dissolved in the same buffer used for electrophoresis, optionally adding EtBr, then pouring the solution into a horizontal casting tray. Multiple gel combs can be used to form rows of wells. After cooling and polymerization, the gel is loaded in a horizontal electrophoresis apparatus and covered with buffer in a single chamber. Wells are loaded and electrophoresis performed as described above. Agarose gels have a large pore size. Agarose gels with a concentration of 1% are used to separate DNA fragments of 1 to 20 kilobases (kb), while higher-concentration gels are useful to separate smaller DNA fragments. Agarose gels are thicker and more stable than polyacrylamide gels but do not provide the same degree of resolution. Agarose is safer than acrylamide but still must be handled and disposed of with care if the gel contains EtBr. Other modified agarose compounds are available that can be mixed in various ratios with standard agarose to increase the resolution of agarose gels. Like acrylamide gels, precast agarose gels are commerically available.

Capillary Electrophoresis

Capillary electrophoresis (CE) is a widely used separation technology for analysis of proteins, peptides, chemicals, natural products, pharmaceuticals, and DNA. Capillary electrophoresis systems are commercially available and generally provide more consistent and standardized results with less time and effort than gel electrophoresis. Using CE, DNA fragments are rapidly separated with a high-voltage gradient, because the capillary dissipates heat quickly. Therefore, one CE run takes approximately 0.5 hour or less, and if eight or 16 capillaries are run simultaneously, the process reduces the time from standard electrophoresis, which requires 3 to 4 hours. This is a significant time saving in the clinical laboratory for applications such as sequencing. CE enables more standardized results, maximization of workforce efficiency, increased productivity and throughput, and the potential for error reduction. CE also uses smaller sample volumes.

In CE, electrophoretic separation takes place in a capillary tube ranging in length from 25 to 100 cm and approximately 50 to 75 |im in diameter. Most capillary tubes are made of glass (silica) walls that often are covered with an external polyimide coating. Acid silanol groups impart a negative charge on the internal wall of the capillary. A low-viscosity acrylamide-based flowable polymer acts as the electrolyte solution and sieving matrix within the silica capillary and is responsible for the conductivity of current through the capillary. Polymer concentration affects the pore size and movement characteristics of the DNA.

A small section of the capillary coating is removed at one end of the capillary to create a detection window. The detection window is optically aligned with the detection system of the instrument. The detection system often includes either a diode or argon laser combined with a charge-coupled device (CCD) camera or filter wheel and photomultiplier tube. The opposite end of the capillary and electrode is used for the injection of the sample. Sample injection in CE is frequently performed by electrokinetic injection. In electrokinetic injection, the capillary and electrode are moved into the sample well. The sample enters the capillary when a voltage of 2 to 5 kV is applied for approximately 5 to 15 seconds. The voltage causes sample ions including DNA to migrate electrophoretically into the capillary in a flat flow profile. Electrokinetic injection produces increased resolution compared to hydrody-namic injection, which produces a laminar (curved) flow profile. After the injection, the capillary and electrode are returned to a buffer reservoir for the separation. The DNA fragments separate by size during migration through the capillary and are detected through the window at the far end of the capillary.

In the clinical molecular laboratory, DNA sequencing and DNA fragment sizing or quantitation are the most common applications performed on CE instruments. One negative aspect of CE as opposed to older polyacrylamide gel technology is that CE is more sensitive to contaminants and DNA concentration. DNA, being negatively charged, migrates into the capillary when voltage is applied. If there are any other charged particles in the sample, they also are injected into the capillary. For example, salt is an ionic competitor. If salt is present, the fluorescent signal intensity of the sample will be greatly reduced because of ionic competition during the brief injection. Proper sample preparation is therefore a key to successful CE.

After a postreaction purification step, if needed for the specific CE application, DNA samples are resuspended in a sample loading solution. High-quality deionized for-mamide often is used as the sample loading solution. If DNA is denatured prior to CE, the formamide maintains the denatured state of DNA and provides a very stable environment for fluorescent dyes. Following the postreaction purification and resuspension of products, the samples are ready for analysis on the CE instrument. The fragments are injected into the capillary and detected by laser-induced florescence, and data are generated for analysis using software supplied by the manufacturer for different CE applications.

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