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 degrees C) based on polyethyleneoxide (PEO) to separate DNA fragments up to 40 kbp. The matrix contained a mixture of 0.5% PEO (Mn 10(6)) to separate fragments up to 1.5 kbp, combined with 0.1% PEO (Mn 8 x 10(6)) 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.
Madabhushi
R S
RS
Molecular Dynamics, Inc., Sunnyvale, CA 94086, USA.
Vainer
M
M
Dolnik
V
V
Enad
S
S
Barker
D L
DL
Harris
D W
DW
Mansfield
E S
ES
D004396:Coloring Agents; D004247:DNA / Q000302:isolation & purification*; D019075:Electrophoresis, Capillary / Q000379:methods*; D011092:Polyethylene Glycols; D014783:Viscosity
D004396:Coloring Agents; D011092:Polyethylene Glycols; D004247:DNA