THE EFFECT OF DNA CONCENTRATION ON MOBILITY IN PULSED FIELD GEL-ELECTROPHORESIS

The effect of DNA concentration on pulsed field gel electrophoretic mobility was studied for human genomic DNA prepared in agarose inserts at 8-800-mu-g/ml and digested to completion with Not I. An eighth of each 100-mu-l insert was used to produce DNA loads of 0.1 to 10-mu-g per lane. The mobility of single copy restriction fragments, as detected by hybridization, was largely concentration independent when DNA concentrations were 80-mu-g/ml or less. However, at DNA concentrations of 200-mu-g/ml and greater, dramatic effects of DNA concentration are evident. In the worst case, at 800-mu-g/ml, the apparent size of a DNA fragment is almost 2.5 times its true size. At constant DNA concentrations, increasing the DNA mass loads by loading larger insert slices had no further effect on DNA electrophoretic mobility, although the bands were broader for bigger insert slices. Thus, for precise and accurate sizing in pulsed field gel electrophoresis the DNA concentration in agarose inserts should not be greater than 80-mu-g/ml (10(7) diploid human cells/ml agarose insert).