Sieving Matrix Selection

The charge density of nucleic acids is uniform and constant for nucleic acid molecules of varying length. Therefore, they typically migrate at the same velocity in response to an electrical potential in free solution. Capillary electrophoretic (CE) separation of nucleic acid species requires addition of a sieving matrix to the background electrolyte to retard the analytes in proportion to their molecular size (1 ). This can be achieved by casting a crosslinked gel in the capillary, or by incorporating a linear gel material or polymer in the background electrolyte. Both of these approaches have been applied successfully to the separation of nucleic acid species ranging from short, single-stranded oligonucleotides to dsDNA of several kilobase lengths. The selection of the appropriate sieving matrix will depend upon the size of the molecules that are to be separated, the resolution required, and the capabilities of the analytical instrumentation. This chapter will review the various sieving matrices which have been employed for CE separation of nucleic acids, identify the appropriate matrix for specific applications, and provide practical suggestions for preparation and use of sieving media for CE.