HIGH SPEED ELECTROPHORESIS IN A SOLID SUBSTRATE

A METHOD FOR THE HIGH SPEED ELECTROPHORETIC SEPARATION OF NESTED SETS OF NUCLEIC ACID FRAGMENTS IN A SOLID (I.E., NONGEL) SUBSTRATE IS BEING DEVELOPED. THE TECHNIQUE EMPLOYS THIN CELLULOSE MEMBRANES AS THE SUPPORTS FOR THE SEPARATION OF SEQUENCING FRAGMENTS IN AN ELECTRIC FIELD. NUCLEIC ACID (DNA, RNA) FRAGMENTS ARE DRIVEN THROUGH PORES IN A CELLULOSE MATRIX. THE CELLULOSE MATRIX PORE DIAMETERS ARE ON THE ORDER OF THE DIAMETER OF DOUBLE-STRANDED DNA. THIS IS BEING ACCOMPLISHED BY CAUSING THE DNA FRAGMENTS TO MIGRATE BY REPTATING (THREADING "END-ON") THROUGH THE CELLULOSE MATRIX PORES. FRAGMENTS BECOME ORIENTED END-ON BEFORE THEY ENTER THE CELLULOSE MATRIX BY WAY OF A SHORT, STEEP PORE GRADIENT CONTIGUOUS WITH THE TOP OF THE CELLULOSE MEMBRANE. A SIMILAR APPROACH, USING PORE-LIMITING CONCENTRATIONS OF POLYACRYLAMIDE ELECTROPHORESIS, HAS BEEN SHOWN TO RESULT IN THE HIGH RESOLUTION SEPARATION OF DNA RESTRICTION FRAGMENTS IN LESS THAN 1 MINUTE. IT IS ANTICIPATED THAT THIS LEVEL OF PERFORMANCE CAN BE DUPLICATED BY EMPLOYING SIMILAR PORE DIAMETERS IN A CELLULOSE MATRIX. ADDITIONAL ADVANTAGES THAT COULD BE AFFORDED BY THE USE OF CELLULOSE MEMBRANES FOR ELECTROPHORETIC SEPARATIONS INCLUDE THEIR COMMERCIAL AVAILABILITY AND POTENTIAL FOR LONG-TERM STORAGE; UNLIKE THE NEUROTOXIC ACRYLAMIDE MONOMER AND CROSSLINKER, CELLULOSE IS NONTOXIC. IN PHASE I RAPID, REPTATIVE ELECTROPHORETIC SEPARATION OF SEQUENCING-SIZED FRAGMENTS (I.E., IN THE RANGE OF 10 TO 300 BASE-PAIRS) ARE BEING OPTIMIZED BY USING THIN CELLULOSE MEMBRANES. STUDIES ARE BEING UNDERTAKEN TO DETERMINE THE OPTIMUM (1) METHOD FOR SAMPLE LOADING; (2) BUFFER CONDITIONS; (3) ELECTRIC FIELD PARAMETERS, INCLUDING TESTING OF CONTOUR-CLAMPED HOMOGENEOUS FIELD CONDITIONS; AND (4) STORAGE CONDITIONS, SHELF LIFE, AND REUSABILITY OF CELLULOSIC MEMBRANES. IN PHASE II, HIGH SPEED SEQUENCE ANALYSIS IN LARGE FORMAT CELLULOSE MEMBRANES WILL BE OPTIMIZED BY USING VARIOUS APPROACHES TO THE GENERATION OF SEQUENCING FRAGMENTS. THE GOAL OF PHASE II WILL BE THE DEVELOPMENT OF KITS FOR SPECIFIC APPLICATIONS.