Discontinuous electrophoresis of DNA: adjusting DNA mobility by trailing ion net mobility.
The use of a discontinuous buffer system, where a moving boundary separates ions of like charge but different ionic mobilities, for DNA electrophoresis may hold advantages over continuous zone electrophoresis in terms of resolution and electrophoresis time. Discontinuous buffer systems with calculated trailing ion net mobility were used to evaluate DNA mobility on gels of a constant pore size. Standard double-stranded DNA ladders and dideoxy sequencing ladders were electrophoresed on open-faced gels and standard sequencing gels, respectively. Trailing ion net mobility was systematically varied, while the leading ion mobility and concentration were kept constant. Decreasing trailing ion net mobility from 2.17 x 10(-4) to 0.59 x 10(-4) cm2 V-1s-1 generally led to increased DNA migration on both native and denaturing gels, allowing resolution of higher molecular weight DNAs with decreased electrophoresis time. However, on native open-faced gels, net trailing ion mobilities between 1.38 x 10(-4) and 1.76 x 10(-4) cm2 V-1s-1 had no differential effects for a 10-cm separation and kept DNAs smaller than approximately 75 bp stacked in the moving boundary and clearly resolved DNA between 100 and 600 bp. These results indicate that various DNA size ranges can be separated in short time periods by adjusting the net mobility of the trailing ion.
Doktycz
M J
MJ
Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-8077.
D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D002021:Buffers; D004247:DNA / Q000032:analysis* / Q000302:isolation & purification; D004277:DNA, Single-Stranded / Q000032:analysis* / Q000302:isolation & purification; D004586:Electrophoresis / Q000379:methods; D005782:Gels; D017422:Sequence Analysis, DNA / Q000379:methods
D000596:Amino Acids; D002021:Buffers; D004277:DNA, Single-Stranded; D005782:Gels; D004247:DNA