Capillary Gel Electrophoresis

Abstract

There is a great deal of interest in analytical biochemistry in the separation and identification of biologically important polymers, such as DNA protein and complex carbohydrate molecules (1,2). For relatively short single-stranded DNA units (i.e., oligonucleotides) and carbohydrate molecules, there is a need to separate by a single base difference (for DNA sequencing) (3) or even for identical chain length with a different sequence (identification of primers, probes, and antisense DNA molecules) (3,4). For the double-stranded DNA molecules, there is an interest to analyze and identify DNA molecules in the form of restriction fragments or PCR products. Using various types of sieving media allows us to do these kinds of separations. In capillary gel electrophoresis, crosslinked or noncrosslinked sieving matrices can be employed (5–7). The crosslinked gels, i.e., chemical gels, have a well-defined pore size. Noncrosslinked, or so-called physical gels, have a dynamic pore structure. This major difference provides the noncrosslinked linear polymer networks with much higher flexibility when compared to the crosslinked gels. One can operate at high temperatures (up to 50–70°C) while applying extremely high field strengths (up to 10³ V/cm range) without any damage to the linear polymer network formulations (8. It is important to note that the crosslinked gels are not usable under such extreme conditions (9). The other main advantage of the linear polymer network system is that it can be easily replaced in the capillary column by simply rinsing the gel matrix through the capillary by pressure or vacuum. Therefore, if the column becomes contaminated, the gel is easily replaced extending the lifetime of the system. Employing the replaceable concept, there is a possibility of the use of pressure injection compared to the crosslinked gels where electrokinetic injection mode is the only possibility (10). It is important to note that in addition to convenience, pressure injection permits quantitative analysis.