Applications of short-chain polydimethylacrylamide as sieving medium for the electrophoretic separation of DNA fragments and mutation analysis in uncoated capillaries.
In capillary electrophoresis (CE), separation of DNA fragments is usually performed in covalently coated capillaries. Recent studies have demonstrated that certain polymers form a dynamic coating on the inner surface of the capillary, thereby suppressing the electroosmotic flow and DNA-capillary wall interactions. We developed a simple method for the synthesis of short-chain polydimethylacrylamide (PDMA) using isopropanol as a chain transfer agent. Capillary (<75 microm internal diameter) filling and replacement of this low-viscosity (14 cP at 4% PDMA) self-coating medium were easily carried out by commercial CE instruments. Using PDMA and uncoated capillaries, we first examined the separation of phi X174 HaeIII DNA digests and observed that the stability of the dynamic coating was markedly better at pH 7.8 than at pH 8.3. At this lower pH and nondenaturing conditions, high resolution of the phi X174 HaeIII DNA digests was obtained for more than 850 injections in the same capillary. We then exploited this sieving medium in CE using multiple approaches for mutation analysis of clinical DNA samples including separation of restriction enzyme cleavage products, analysis of single strand conformation polymorphisms, and simultaneous detection of several mutations using multiplex allele-specific PCR amplification. Our results demonstrate that CE in uncoated capillaries using PDMA as sieving medium is a simple, versatile, and reliable strategy for separation and mutation analysis of clinical DNA samples.
Ren
J
J
Department of Pharmacology, University of Bergen, Armauer Hansens Hus, N-5021 Bergen, Norway. jicun.ren@farm.uib.no
Ulvik
A
A
Refsum
H
H
Ueland
P M
PM
D000180:Acrylic Resins / Q000138:chemical synthesis / Q000737:chemistry; D010584:Bacteriophage phi X 174 / Q000737:chemistry; D001483:Base Sequence; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification*; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000302:isolation & purification; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018807:Polymorphism, Single-Stranded Conformational; D000099166:Polyacrylamides
D000180:Acrylic Resins; D004247:DNA; D017931:DNA Primers; D004279:DNA, Viral; D000099166:Polyacrylamides