DNA SEQUENCING BY HYBRIDIZATION ON SURFACES: DEVELOPMENT OF ULTRA-SENSITIVE TWO-DIMENSIONAL DETECTORS

THE NEED TO MAP AND SEQUENCE DNA IN THE HUMAN GENOME IS OF CRUCIAL IMPORTANCE FOR A BETTER UNDERSTANDING OF GENETICS AND DISEASE PROCESSES. CURRENT DNA SEQUENCING PROCEDURES ALMOST UNIVERSALLY REQUIRE GEL ELECTROPHORESIS OF DNA FRAGMENTS, AND SEVERAL LABORATORIES ARE DEVISING PROCEDURES TO AUTOMATE AS MANY STEPS OF THIS PROCESS AS POSSIBLE. SEQUENCING BY HYBRIDIZATION (SBH) PROPOSED BY DRMANAC ET AL. DOES NOT EMPLOY ELECTROPHORESIS, BUT INSTEAD UTILISESAN ARRAY OF SHORT OLIGONUCLEOTIDES REPRESENTING ALL POSSIBLESEQUENCES IN DNA WHICH HYBRIDIZE TO THE UNKNOWN FRAGMENT OF DNA TO BE SEQUENCED. THE SEQUENCE OF THE UNKNOWN FRAGMENT CAN BE RECONSTRUCTED BY COMPUTER METHODS. ATTACHING THE ARRAY TO A SURFACE IN A PRE-DETERMINED PATTERN, THE DNA SEQUENCING MATRIX, LEADS TO A HIGHLY EFFICIENT MEANS OF SEQUENCING LARGE PORTIONS OF GENOMIC DNA. THE DEVELOPMENT OF THIS TECHNIQUE WILL REQUIRE COST-EFFECTIVE METHODS FOR THE DETECTION OF SURFACE-BOUND DNA WITH HIGH SENSITIVITY, SELECTIVITY, SPATIAL RESOLUTION, AND HIGH ANALYSIS RATE OF THE MULTIPLE SITES WITHIN THE ARRAY OF OLIGONUCLEOTIDES. DURING PHASE I WE WILL EXPERIMENTALLY EVALUATE THE MOST PROMISING DETECTION TECHNIQUES FOR POSITIVELY IDENTIFYING HYBRIDIZED AND UNHYBRIDIZED SITES ON A DNA SEQUENCING MATRIX. TEST MATRICES ONTO WHICH 8-MER (OR LARGER) OLIGONUCLEOTIDE SEQUENCES HAVE BEEN ATTACHED WILL BE ANALYZED BY SPUTTER-INITIATED RESONANCE IONIZATION SPECTROSCOPY (SIRIS), LASER ATOMIZATION RESONANCE IONIZATIONSPECTROSCOPY (LARIS), AND LASER-EXCITED FLUORESCENCE (LEF). THE EFFORT IS DIRECTED TOWARD DEFINING AN OPTIMAL DETECTION SYSTEM FOR ACHIEVING MINIMIZATION OF MATRIX SIZE AND ANALYSIS TIME, WHILE MAINTAINING HIGH ACCURACY. RECOMMENDATIONS WILL BE MADE FOR THE MOST FRUITFUL DNA SEQUENCING DIRECTIONS DURING PHASE II, AND A DETECTION SYSTEM DEDICATED TO THE STUDY OF DNA SBH WILL BE SPECIFIED.