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Electrophoresis for Sequencing and Sizing

Electrophoresis in the CZE mode takes place in an open tube and in a free solution without any separation matrix in the capillary. The separation is based on the mass/charge ratio of the analytes. It is appropriate for the separation of nucleosides and nucleotides. It is not well suited for medium to large oligonucleotides, because their mass/charge ratio tend to be smaller. The use of a separation matrix becomes necessary for these species. Various capillary systems, including bare fused silica capillaries and surface-coated capillaries, have been used in CZE. [Pg.365]

Linear/non-cross-linked polymers are an alternative to cross-linked polymers, where the separation can be carried out in the CGE format. These polymer solutions are made of hydrophilic polymers dissolved in an appropriate buffer. They have relatively lower viscosity than the cross-linked polymers. They can be pumped out of the capillary at the conclusion of a run, thus allowing a fresh separation media to be used for each analysis. In case of cross-linked polymers, once the separation medium has degraded, the entire capillary must be replaced. This can be tedious for example, it may require realignment of the optical system with the narrow-bore capillary. The linear polymer solutions can withstand temperatures up to 70° C and high field strengths up to 1000 V/cm. [Pg.366]

The mechanism of separation with linear polymers is as follows. At a certain polymer concentration known as the entanglement threshold, the individual polymer strands begin to interact with each other, leading to a meshlike structure within the capillary. This allows DNA separation to take place. Many of the common polymers are cellulose derivatives, such as hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, and methylcellulose. Other applicable polymers include linear polyacrylamide, polyethylene oxide, agarose, polyvinyl pyrrolidone, and poly-N. Ar-dimethylacrylamide. High-resolution separation up to 12,000 bp has been reported using entangled polymer solutions. [Pg.366]


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