DNA separation by microchip electrophoresis using low-viscosity hydroxypropylmethylcellulose-50 solutions enhanced by polyhydroxy compounds.
Low-viscosity polymer solutions have potential for double-stranded (ds) DNA separations in micrototal analysis systems (micro-TAS). In this paper, we report dilute, low-viscosity hydroxypropylmethylcellulose-50 (HPMC-50, 11.5 kDa) solutions containing polyhydroxy additives as separation media. Predominant operational variables, such as applied electric field strength, fluorescent intercalator (YOPro-1) concentration, polymer concentration, and additive concentration, are thoroughly investigated. Fast (within 170 s) and excellent separation of DNA restriction fragments ranging in size from 72 to 1353 base pairs (bp) is achieved in a 30 mm length channel of polymethylmethacrylate (PMMA) microchips at an electric field strength of 300 V/cm, by introducing 8% mannitol, 8% glucose or 10% glycerol additives into a 2% HPMC-50/1 x Tris-borate-EDTA (TBE) solution. The low-viscosity (40 cP) matrix formulation provides both coating of the microchannels and separation of DNA in one step. The performance in the solution surpasses that in highly concentrated HPMC-50 solution. In addition, separation using 1xTris-EDTA buffer in the 2% HPMC-50 matrix containing polyhydroxy additives also exhibits a notably increased performance. This is presumably due to formation of hydrogen-bonding interactions of polyhydroxy additives with HPMC-50 matrix and DNA so as to increase the coupling interactions between matrix and DNA molecules during electrophoresis. The result reflects that boric acid is not a prerequisite in polyhydroxy-enhanced HPMC-50 solution for separation.
Xu
Feng
F
Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, The University of Tokushima, Shomachi, Tokushima 770-8585, Japan. fengxu64@hotmail.com
Jabasini
Mohammad
M
Baba
Yoshinobu
Y
D010584:Bacteriophage phi X 174 / Q000235:genetics; D004247:DNA / Q000302:isolation & purification*; D004279:DNA, Viral / Q000302:isolation & purification; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D005947:Glucose / Q000737:chemistry; D005990:Glycerol / Q000737:chemistry; D065347:Hypromellose Derivatives; D008353:Mannitol / Q000737:chemistry; D008747:Methylcellulose / Q000031:analogs & derivatives*; D008904:Miniaturization; D014783:Viscosity
D004279:DNA, Viral; D065347:Hypromellose Derivatives; D008353:Mannitol; D008747:Methylcellulose; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C024403:GGCC-specific type II deoxyribonucleases; D005947:Glucose; D005990:Glycerol