Effect of total percent polyacrylamide in capillary gel electrophoresis for DNA sequencing of short fragments. A phenomenological model.
Polyacrylamide capillary gels were prepared with constant (5% C) cross-linker concentration and with total acrylamide concentration ranging from 2.5 to 6% T. At each acrylamide concentration, peak spacing was constant for DNA sequencing fragments ranging from 25 to 250 nucleotides in length. Peak spacing increased linearly with the total acrylamide concentration. The intercept of the retention time vs. fragment length plot was independent of % T. Ferguson plots were constructed for short DNA fragments; the polyacrylamide pore size falls in the 2.5 to 3.5 nm range for the gels studied. Theoretical plate count is independent of total acrylamide concentration; longitudinal diffusion, and not thermal gradients, limit the plate count. A phenomenological model is presented that predicts retention time, plate count, and resolution for sequencing fragments ranging in size from 25 to 250 bases and gels that range from 2.5 to 6% total acrylamide.
Harke
H R
HR
Department of Chemistry, University of Alberta, Edmonton, Canada.
Bay
S
S
Zhang
J Z
JZ
Rocheleau
M J
MJ
Dovichi
N J
NJ
D000180:Acrylic Resins / Q000737:chemistry*; D001483:Base Sequence; D004247:DNA / Q000032:analysis*; D004058:Diffusion; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation*; D008962:Models, Theoretical; D000099166:Polyacrylamides
D000180:Acrylic Resins; D004247:DNA; D000099166:Polyacrylamides