OPTIMIZATION OF ELECTROPHORESIS GELS FOR RIS ANALYSIS

GIVEN A FAST, SENSITIVE DETECTION METHOD, A LARGE NUMBER OF STABLE ISOTOPES COULD BE USED TO LABEL DNA, THEREBY MULTIPLEXING THE SEPARATION PROCESS, AND PROVIDING A NEW, MUCH FASTER PROCEDURE FOR LOCALIZING DNA AFTER ELECTROPHORESIS. SPUTTER-INITIATED RESONANCE IONIZATION SPECTROSCOPY (SIRIS) CAN BE USED TO MEASURE SUBATTOMOLE QUANTITIES OF ISOTOPE-LABELED DNA WITH EXCELLENT LATERAL AND MASS RESOLUTION. SIRIS ANALYSIS HAS THE POTENTIAL OF ORDERS-OF-MAGNITUDE IMPROVEMENT IN THE SPEED OF DNA SEQUENCING, BUT REQUIRES ELECTROPHORESIS GELS IN WHICH THE SEPARATED DNA FRAGMENTS ARE CONCENTRATED AT THE GEL SURFACE AND IN BANDS OF ABOUT 100 FM WIDTH. THE OBJECTIVE IS TO DEVELOP ANALYSIS METHODS FOR ELECTROPHORESIS GELS THAT CAN LOCALIZE SUBFEMTOMOLE CONCENTRATIONS OF ISOTOPE-LABELED DNA WITH MICROMETER LATERAL AND NANOMETER DEPTH RESOLUTION LEADING TO IMPROVED ELECTROPHORESIS MATERIALS AND PROCESSES THAT BEST MATCH THE ULTRAHIGH ELEMENT SENSITIVITY AVAILABLE WITH SIRIS. WE WILL OPTIMIZE SIRIS TO DETERMINE THE SPATIAL DISTRIBUTION OF ISOTOPE-LABELED DNA BANDS IN ULTRATHIN PAGE SAMPLES AND EXPLOIT OPTIMIZATION OF GEL PARAMETERS SUCH AS CASTING PROCEDURES, THICKNESS, MATRIX MODIFIERS, TEMPERATURE SHRINKING RATIOS, DRYING PROCESSES LEAD TO HIGH RESOLUTION GEL ELECTROPHORESIS WITH SURFACE ENHANCED DNA DISTRIBUTION BY THE END OF PHASE II.