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Proceedings Paper

Linking electrophoretic resolution with experimental conditions
Author(s): John Clark Sutherland
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Paper Abstract

Previously, we described a quantitative measure of electrophoretic resolution called resolving power that can be computed for individual bands of DNA separated by electrophoresis. An alternate approach is to determined analytical functions, based on a few experimentally determined parameters, that describe the resolving power of a particular electrophoretic system for al of the length classes of resolving power of a particular electrophoretic system for all of the length classes of molecules that are separated. Such analytical functions have been obtained for single-stranded DNA separated in a polyacrylamide gel and detected at a fixed distance from the origin of electrophoresis. Six experimentally determined constants are required to describe the analytical function for resolving power in this system: four constants describe the mobility of DNA in the gel as a function of molecular length, and two describe the width of the bands as a function of the length of molecular length. One of our goals is to develop systematic methods of improving the resolving power of longer molecules, and hence extending the number of bases that can be determined in a single sequencing experiment. Our approach is to determine how the small number of parameters that describe resolving power depend on experimentally controllably conditions. Knowing such relationships should permit systematic selection of combinations of experimental conditions that improve resolving power. Here, we show the relationship between the four parameters describing DNA mobility as a function of molecular length for systematic variations of gel composition and electric field strength. The data set employed is for double-stranded DNA separated in agarose gels, but the principles are similar to those encountered in DNA sequencing studies.

Paper Details

Date Published: 22 May 1997
PDF: 14 pages
Proc. SPIE 2985, Ultrasensitive Biochemical Diagnostics II, (22 May 1997); doi: 10.1117/12.274367
Show Author Affiliations
John Clark Sutherland, Brookhaven National Lab. (United States)

Published in SPIE Proceedings Vol. 2985:
Ultrasensitive Biochemical Diagnostics II
Gerald E. Cohn; Gerald E. Cohn; Steven A. Soper, Editor(s)

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