DIRECT SEQUENCING OF LARGE RECOMBINANT MOLECULES

THIS PROJECT ADDRESSES THE NEED FOR EFFICIENT METHODS TO RAPIDLY DETERMINE THE DNA SEQUENCE OF YEAST ARTIFICIAL CHROMOSOME (YAC) OR COSMID CLONES WITHOUT SUBCLONING SMALL FRAGMENTS. THE METHOD COMBINES MULTIPLEX DNA SEQUENCING WITH LIGASE-MEDIATED POLYMERASE CHAIN REACTION (PCR) AMPLIFICATION OF SPECIFIC RESTRICTION FRAGMENTS. PURIFIED YAC OR COSMID DNA IS DIGESTED WITH TWO DIFFERENT RESTRICTION ENZYMES TO GENERATE FRAGMENTS WITH ONE BLUNT ENDAND ONE AMBIGUOUS 4-BASE 5'-PROTRUDING END. SYNTHETIC OLIGONUCLEOTIDES CONTAINING MULTIPLEX TAG SEQUENCES ARE SPECIFICALLY LIGATED TO THESE ENDS, AND PRIMERS HOMOLOGOUS TO THE TAGS ARE USED TO AMPLIFY THE FRAGMENTS BY PCR. PRODUCTS OF THE PCR REACTIONS ARE POOLED, AND CHEMICAL SEQUENCING REACTIONS ARE PERFORMED. THE SAMPLES ARE FRACTIONATED ON SEQUENCING GELS, ELECTROBLOTTED, AND INDIVIDUAL SEQUENCE LADDERS VISUALIZED ON AUTORADIOGRAMS AFTER SEQUENTIAL HYBRIDIZATION USING MULTIPLEX PROBES. WITH APPROPRIATE SELECTION OF RESTRICTIONENZYMES, THERE IS A HIGH PROBABILITY THAT ANY PARTICULAR 4-BASE END WILL OCCUR ONLY ONCE IN THE STARTING CLONE. EXTENSIVE OVERLAPS CAN BE GENERATED BY USING SEVERAL SETS OFINTERRELATED DOUBLE RESTRICTION DIGESTS. PHASE I STUDIES WILL FOCUS PRIMARILY ON TECHNICAL ASPECTS OF THE LIGATION AND PCR REACTIONS. A SET OF MULTIPLEX SEQUENCING REACTIONS WILL BE PERFORMED, AND THE APPLICABILITY OF THE METHOD WILL BE EVALUATED.