DEVELOPMENT OF MICRON TO SUBMICRON THICKNESS ELECTROPHORESISGELS TO OPTIMIZE RESOLUTION IN DNA SEQUENCING USING RIS

TYPICALLY, DNA SEQUENCING IS ACCOMPLISHED USING ELECTROPHORESIS GELS SEVERAL HUNDRED MICRONS THICK, BECAUSE THESE GELS TRANSPORT THE NECESSARY CONCENTRATION OF DNA FRAGMENT MARKERS (FLUORESCENT OR RADIOACTIVE), AND BECAUSE THE TECHNOLOGY FOR UTILIZING THESE GELS IS MATURE. RECENT WORK HAS SHOWN THAT BETTER RESOLUTION CAN BE OBTAINED WITH THINNER GELS AND, WITH A CORRESPONDING REDUCTION IN MIGRATION TIME, ADEQUATE FRAGMENT SEPARATION CAN STILL BE OBTAINED. TO BENEFIT FROM THESE IMPROVEMENTS, A FAST, HIGH RESOLUTION, AND SENSITIVE METHOD FOR DETECTING THE MARKERS ON THE FRAGMENTS MUST BE EMPLOYED. SPUTTER-INITIATED RESONANCE IONIZATION SPECTROSCOPY (SIRIS) OFFERS THESE FEATURES AND ALSO OFFERS THE ABILITY TO EMPLOY A SERIES OF STABLE ISOTOPES AS MARKERS. THUS, ALL FOUR NUCLEOTIDES CAN BE IDENTIFIED IN A SINGLE LANE, FURTHER INCREASING THE ANALYSIS SPEED AND REDUCING THE NEED FOR GEL UNIFORMITY. SIRIS CAN BE USED TO MEASURE SUBATTOMOLE QUANTITIES OF ISOTOPE-LABELED DNA WITH EXCELLENT LATERAL AND MASS RESOLUTION, AND HAS THE POTENTIAL OF ORDERS-OF-MAGNITUDE IMPROVEMENT IN THE SPEED OF DNA SEQUENCING. BECAUSE ONLY THE MARKERS IN THE FIRST FEW MOLECULAR LAYERS OF THE GEL AREDETECTED, ELECTROPHORESIS GELS ARE REQUIRED IN WHICH THE SEPARATED DNA FRAGMENTS ARE CONCENTRATED AT THE GEL SURFACE TO OBTAIN HIGH SENSITIVITY. THE OBJECTIVES OF THIS PHASE I PROJECT ARE (1) TO DEVELOP A METHOD FOR PRODUCING MICROTHIN ELECTROPHORESIS GELS WITH SUBFEMTOMOLE CONCENTRATIONS OF ISOTPE-LABELED DNA AT THE SURFACE AND (2) TO CHARACTERIZE AND DEMONSTRATE THE PERFORMANCE OF THE GEL USING SIRIS.