Resolving power: a quantitative measure of electrophoretic resolution.
Resolving power is a quantitative measure of the ability of an electrophoretic system to separate DNA (and other) molecules of similar size. It is a dimensionless quantity, and hence facilitates comparison of the performance of electrophoretic systems that operate very differently. Resolving power can be determined as a function of molecular length from experimental data consisting of a series of completely resolved bands on a gel or blot; closely spaced bands are not required. We discuss factors such as the mass of DNA in a particular band and the spatial resolution of the system used to image the distribution of DNA on a gel or blot that, while not an intrinsic part of the electrophoretic system, may influence the observed resolving power. We derive an empirical global dispersion function that applies both to images of gels obtained after a fixed time of electrophoresis of all the samples and to images obtained as each species reaches a detector located at a fixed distance from the starting well. We use this dispersion function to show that the improvement in resolving power produced by extending the time or distance of electrophoresis in a static, uniform electric field asymptotically approaches a limiting value that is a function of the length of the DNA. When plotted as a function of molecular length, this limiting value defines an envelope that characterizes the intrinsic limits of performance of a particular electrophoretic system (e.g., electric field strength, gel type and concentration, buffer, temperature). Comparing the resolving power of static field agarose gel electrophoresis as routinely practiced for separating DNA molecules from 10(3) to 10(5) bp long with other electrophoretic schemes suggests that significant improvements should be achievable.
Ribeiro
E A
EA
Biology Department, Brookhaven National Laboratory, Upton, New York 11973.
Sutherland
J C
JC
D017123:Bacteriophage T7 / Q000737:chemistry / Q000235:genetics; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D004279:DNA, Viral / Q000302:isolation & purification*; D004586:Electrophoresis / Q000379:methods*; D004587:Electrophoresis, Agar Gel / Q000379:methods; D007091:Image Processing, Computer-Assisted; D008432:Mathematical Computing; D008970:Molecular Weight; D012680:Sensitivity and Specificity
D004279:DNA, Viral