A method for the high speed electrophoretic separation ofnested sets of nucleic acid fragments in a solid (i.e., nongel)substrate is being developed. The technique employs thin cellulosemembranes as the supports for the separation of sequencingfragments in an electric field. Nucleic acid (DNA, RNA) fragmentsare driven through pores in a cellulose matrix. The cellulosematrix pore diameters are on the order of the diameter ofdouble-stranded DNA. This is being accomplished by causing the DNAfragments to migrate by restating (threading "end-on") through thecellulose matrix pores. Fragments become oriented end-on beforethey enter the cellulose matrix by way of a short, steep poregradient contiguous with the top of the cellulose membrane. Asimilar approach, using pore-limiting concentrations ofpolyacrylamide electrophoresis, has been shown to result in thehigh resolution separation of DNA restriction fragments in lessthan 1 minute. It is anticipated that this level of performancecan be duplicated by employing similar pore diameters in acellulose matrix. Additional advantages that could be afforded bythe use of cellulose membranes for electrophoretic separationsinclude their commercial availability and potential forlong-term storage; unlike the neurotoxic acrylamide monomer and crosslinker, cellulose is nontoxic. In Phase I rapid, repetativeelectrophoretic separation of sequencing-sized fragments (i.e., inthe range of 10 to 300 basepairs) are being optimized by using thincellulose membranes. Studies are being undertaken to determine theoptimum (1) method for sample loading; (2) buffer conditions; (3)electric field parameters, including testing of contour-clampedhomogeneous field conditions; and (4) storage conditions, shelflife, and reusability of cellulosic membranes. In Phase II, highspeed sequence analysis in large format cellulose membranes will beoptimized by using various approaches to the generation 0sequencing fragments. The goal of Phase II will be the developmentof kits for specific applications.Anticipated Results/Potential Commercial Applications as described by the awardee:The potential commercial application of the newtechnology derives from two sources: (1) the marked increase inthroughput afforded by the employment of reptation (end-onmigration) for the separation of sequencing fragments (results frompilot pore-limiting studies suggest that as much as a ten-foldincrease in throughput can be expected from this approach); and (2)whereas polyacrylamide gels for sequence analysis are typicallyprepared in the laboratory by the enduser from reagents that arenotoriously neurotoxic, cellulose membranes are readily available, easily stored, and will likely be reusable. These improvementstranslate directly into commercial advantages over conventionalmethods.
