Fast separation of DNA sequencing fragments in highly alkaline solutions of linear polyacrylamide using electrophoresis in bare silica capillaries.
An optimized procedure for the fast separation of DNA sequencing fragments in short bare fused-silica capillaries filled with highly alkaline solutions of replaceable linear polyacrylamide is presented. High denaturing abilities of the separation media at pH values over 12 are the main reason for their applications in analyses of ssDNA fragments. Moreover, the alkaline solutions of polyacrylamide provide other advantageous properties: three times higher electrophoretic mobility of ssDNA fragments in comparison to those in urea, negligibly low electroosmotic flow in uncoated capillaries, and an adequate stability to a fast alkaline hydrolysis. The separation power of this procedure is enhanced strongly by using monocarboxy poly(ethylene glycol), a terminator for transient isotachophoresis, which eliminates the electromigration dispersion. A high separation efficiency of our system enables to reduce analysis time to several minutes by decreasing the effective lengths of capillaries to 7 cm. A special sample introduction by diffusion is successfully applied. The experimental results demonstrate a potential of the alkaline electrolytes for an implementation in diagnostic sequencing practice.
Klepárník
K
K
Institute of Analytical Chemistry, Academy of Sciences of Czech Republic, Brno. klep@iach.cz
Malá
Z
Z
Bocek
P
P
D000180:Acrylic Resins / Q000737:chemistry*; D004058:Diffusion; D004573:Electrolytes; D019075:Electrophoresis, Capillary / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D009691:Nucleic Acid Denaturation; D009838:Oligodeoxyribonucleotides / Q000302:isolation & purification*; D017422:Sequence Analysis, DNA / Q000379:methods*; D012822:Silicon Dioxide; D000099166:Polyacrylamides
D000180:Acrylic Resins; D004573:Electrolytes; D009838:Oligodeoxyribonucleotides; D012822:Silicon Dioxide; D000099166:Polyacrylamides