VERSATILE LOW-VISCOSITY SIEVING MATRICES FOR NONDENATURING DNA SEPARATIONS USING CAPILLARY ARRAY ELECTROPHORESIS

The high-resolution separation of double-stranded DNA (dsDNA) has impo rtant applications in physical mapping strategies and in the analysis of polymerase chain reaction (PCR) products. Although high-resolution separations of dsDNA by capillary electrophoresis (CE) have been repor ted, pulsed fields were required to achieve complete resolution of DNA fragments beyond 23 kilobase pairs (kbp). Here, we report a single fo rmulation to separate a broad range (80 bp - 40 kbp) of DNA fragments without the use of pulsed fields. We used a low-viscosity sieving medi um (ca. 5 cP, at 25 degrees C) based on polyethyleneoxide (PEO) to sep arate DNA fragments up to 40 kbp. The matrix contained a mixture of 0. 5% PEO (M(a) 10(6)) to separate fragments up to 1.5 kbp, combined with 0.1% PEO (M(a) 8 x 10(6)) to separate fragments between 1-40 kbp, wit hin a single run. All PEO matrix formulations tested were compatible w ith a variety of intercalating dyes and with two different capillary w all coating methods. We obtained a detection limit of 25 fg of a 200 b p DNA quantitation standard using Vistra Green in the matrix. Resoluti on was best using short injection times (5 s or less) and low field st rengths (approximately 100 V/cm). Sample runs were complete in 70 min, and use of the capillary array electrophoresis (CAE) system permitted high-throughput DNA analysis. The size range separated is approximate ly 10 times greater than with conventional slab gel separations.