Capillary electrophoresis with glycerol as an additive.

Abstract

Two kinds of semi-liquid separation media are frequently used for the separation of DNA by high performance capillary electrophoresis (CE), namely dynamic or entangled free solution polymer sieving matrix, and dilute liquid-gel solutions. Polymer solutions may be defined in three different functional concentrations: dilute, semidilute, and entangled. In dilute solutions, the polymer molecules are physically separate; as the concentration of polymer increases the molecules begin to interact, and at a critical concentration the polymers begin to physically interact to form an entangled physical network (, , ). These media have the ability to act as size selective sieving agents that partition DNA molecules, but since the media are liquid the injection of fresh media and the removal of expended media are facilitated. The media are typically neutral high molecular weight polymers and polymer sugars, which provide a high uniformity of electrophoretic medium if capillary electroosmotic flow (EOF) effects are suppressed. For the purpose of this review, particular attention will be drawn to entangled free solution capillary electrophoresis (ESCE) using Tris-borate-EDTA (TBE) buffers, with glycerol as an additive. For DNA separations, many different ESCE sieving media and dilute liquid-gel solutions have been used (Table 1): polysaccharide polymers such as, hydroxypropyl cellulose (HPC) (, , ), hydroxypropyl methyl cellulose (HPMC) (, , ), methyl cellulose (,), hydroxyethyl cellulose (HEC) (,,, , , , , ), dextran (,), TreviSol (), semi-liquid agarose (,), and galactomannans (). Nonsaccharide polymers such as linear polyacrylamides and substituted polyacrylamides (,,, , , , , , , , , , , , , , , , ), linear and branched poly(ethylene oxides), and poly vinyl polymers () are also employed for a wide variety of separations purposes. Table 1 Application of Glycerol Containing Buffers for DNA Analysis by CE a Assay Matrix Buffer Detector Comment Ref. dsDNA PCR-RFLP HPMC TBE/glycerol±ethidium UV, LIF Glycerol improves resolution 5,6,46,47,49 dsDNA PCR-RFLP 0.2–1% HEC TBE UV Good resolution 2,4,10,11 STR-PCR PCR-RFLP 0.5% MC 0.5–2.5X TBE TAE YoPro, LIF, no dye, UV Better resolution in TBE 8,9,15 STR-PCR PCR-RFLP 1.25% HEC TBE 7 Murea no dye, UV Good resolution of several STR 14 Supercoil plasmid 0.1–0.3% HEC 0.5–2.5X TBE UV Good resolution 54 dsDNA LCR HPMC TBE/glycerol LIF Good resolution 25 and 50 bp 48 dsDNA PCR-RFLP 0.5% PEO TBE/dyes LIF Good resolution 27,55 dsDNA PCR-RFLP Liquid TBE Better resolution in TBE 20,39 Aragose TAE dsDNA PCR-RFLP Trevisol TAPS UV Good resolution 18 HPA HPMC TBE/glycerol±ethidium UV, LIF Glycerol improves resolution 50 SSCP HPMC TBE/glycerol±ethidium UV. LIF Glycerol stablizes confomers 51 SSCP LPA TBE±glycerol UV Good resolution 17,22,52,57 dsDNA PCR-RFLP LPA TBE±ethidium UV Good resolution 21,24,44 RNA HPMC TBE 7 Murea UV Glycerol diminishes resolution 45 a Some comments on the effects of glycerol and borate on DNA resolution are noted.