The Electrophoretic Separation of DNA Molecules

Conventional gel electrophoresis is based on the principle that negatively charged DNA fragments below 50 kb can be separated when subjected to a unidirectional electric field. The DNA samples are added to wells in an agarose matrix and migrate in the direction of the field at a rate that is inversely proportional to size, i.e., the larger the DNA fragments, the slower their rate of migration in the electric field. However, when DNA molecules are larger than the threshold size of 50 kb, all fragments exhibit size-independent mobilities.1-1-1 This significant practical problem limited the use of electrophoresis for the study of megabase-sized molecules, such as the bacterial chromosomal DNA.

In 1984, Schwartz and Cantor introduced the concept of pulsed field gel electrophoresis by using two alternating electric fields, to separate large DNA fragments (50 to 2000 kb) within agarose gels.[2] Subsequently, a variety of alternative electrophoretic configurations, using currents ''pulsed'' in different directions over controlled time intervals, have been developed. These included orthogonal field alternation gel electrophoresis,1-3-1 vertical alternating field gradient gel electrophoresis,[4] periodic field inversion gel electrophoresis,[5] and contour-clamped homogeneous electric field electrophoresis (CHEF).[6]

In PFGE, chromosomal DNA can be treated with a restriction endonuclease, resulting in DNA fragments that vary in size from 50 to 2000 kb. It is a powerful molecular typing technique for comparing and analyzing bacterial chromosomal DNA fragments. The two most common pulsed field methods used in DNA fingerprinting laboratories are the CHEF and the programmable autonomously controlled electrode gel electrophoresis (PACE).[7] Both systems contain three major components: a power module to generate the electrode voltages and store switching function parameters, a cooling module to keep the temperature at 14°C, and an electrophoresis chamber. The chamber contains 24 horizontal electrodes, some of which are clamped to eliminate DNA lane distortion. The electrodes are arranged in a hexagon, thus providing reorientation angles of 60° or 120° in contrast to traditional orthogonal field alternation gel systems with two perpendicular electrodes. The resolution of PFGE is dramatically affected by the number and configuration of the electrodes used, because these alter the shape of the applied electrical field. For high-resolution separation, the most effective electrode configurations yield angles of more than 110°.[8] In PACE, each electrode's voltage is independently controlled and can generate an unlimited number of electric fields of different voltage gradients, orientations, and intervals sequentially in time. However, the traditional CHEF systems are limited to two alternating electric fields at a fixed reorientation angle. The two leading PFGE technologies, CHEF and PACE, have been combined into a CHEF Mapper system (BioRad Inc.) which is widely used by molecular epidemiologists. It has the advantage of containing protocols embedded on a microchip, thus eliminating trial and error in setting parameters for obtaining good resolution of band patterns.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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