Pulsed-Field Capillary Electrophoresis of Large DNA

Abstract

Over its many years of existence, electrophoresis in slab gels has become a very useful technique in biochemistry and biology. It has been commonly applied to a wide range of problems [1]: mostly to the separation of proteins and nucleic acids and, to a lesser degree, of sugars and other poly electrolytes. Research directed toward the separation of nucleic acids has been driven by the explosion of molecular biology and genomic projects; these activities have been particularly visible during the last several years. Conventional DNA separations were initially restricted to the sizes of up to approximately 20–30 kbp (kilo-base pairs) [2]. The reason for this restriction has been the reptative motion of large DNAs in the direction of an applied electric field, and a subsequent molecular orientation [3–6]. At a large molecular weight, the migration velocity of an oriented DNA chain does not scale with N -1 (where N is the DNA size; for abbreviations, see Chapter 2) and becomes size-independent. In other words, the oriented and stretched DNAs of different sizes migrate with the same velocity and will not separate from each other in the continuous gel electrophoresis.