Versatile low-viscosity sieving matrices for nondenaturing DNA separations using capillary array electrophoresis

The high‐resolution separation of double‐stranded DNA (dsDNA) has important 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 reported, pulsed fields were required to achieve complete resolution of DNA fragments beyond 23 kilobase pairs (kbp). Here, we report a single formulation to separate a broad range (80 bp – 40 kbp) of DNA fragments without the use of pulsed fields. We used a low‐viscosity sieving medium (ca. 5 cP, at 25°C) based on polyethyleneoxide (PEO) to separate DNA fragments up to 40 kbp. The matrix contained a mixture of 0.5% PEO (Ma 106) to separate fragments up to 1.5 kbp, combined with 0.1% PEO (Ma 8 × 106) to separate fragments between 1–40 kbp, within a single run. All PEO matrix formulations tested were compatible with a variety of intercalating dyes and with two different capillary wall coating methods. We obtained a detection limit of 25 fg of a 200 bp DNA quantitation standard using Vistra Green in the matrix. Resolution was best using short injection times (5 s or less) and low field strengths (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 approximately 10 times greater than with conventional slab gel separations.