Hydroxyethylcellulose as an effective polymer network for DNA analysis in uncoated glass microchips: optimization and application to mutation detection via heteroduplex analysis.
The nature of the sieving matrix for DNA fragment separation is of immense importance in capillary and microchip electrophoresis. The chemical nature of the surface of the capillary or microchannel wall is equally as important, particularly with DNA electrophoresis where a substantial electroosmotic flow (EOF) may be detrimental to the separation. Although DNA analysis has been carried out successfully in both coated and uncoated capillaries, analysis of unpurified polymerase chain reaction products has been carried out primarily with covalently coated surfaces, especially with microchip electrophoresis. In this report, double-stranded (ds) DNA fragment analysis using hydroxyethylcellulose (HEC) buffered in 1xTris-borate-EDTA is demonstrated both in uncoated capillaries and in microchips. EOF was suppressed 20% in the presence of 1.5% HEC, and the effectiveness of HEC as a polymer for dsDNA fragment analysis was dependent on the pH, with pH 8.6 being optimal. Using separation efficiency (number of theoretical plates) and resolution to gauge the effectiveness of a variety of polymers for the capillary separation of dsDNA fragments in the size range 60-587bp, HEC was found to be comparable in performance to polydimethylacrylamide (PDMA), and superior to linear polyacrylamide and polyethylene oxide for DNA analysis. With respect to longevity and robust performance, HEC could be used effectively in an uncoated capillary for more than 40 runs and for more than 90 runs (without replenishing the polymer) in an uncoated microchip. Application of the optimized HEC conditions is demonstrated through its ability to facilitate heteroduplex analysis.
Tian
Huijun
H
Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Landers
James P
JP
D002021:Buffers; D002482:Cellulose / Q000031:analogs & derivatives* / Q000737:chemistry*; D004247:DNA / Q000032:analysis*; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D019075:Electrophoresis, Capillary / Q000379:methods*; D005453:Fluorescence; D005898:Glass; D020180:Heteroduplex Analysis / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D008832:Microchemistry / Q000295:instrumentation; D016133:Polymerase Chain Reaction; D011108:Polymers; D015203:Reproducibility of Results; D013499:Surface Properties
D002021:Buffers; D017931:DNA Primers; D011108:Polymers; D002482:Cellulose; C002283:hydroxyethylcellulose; D004247:DNA