COUPLED AMPLIFICATION AND SEQUENCING OF DNA

THE OBJECTIVE OF THIS PROJECT IS THE COMMERCIALIZATION OF A METHOD FOR THE COUPLED AMPLIFICATION AND SEQUENCING ("CAS") OF DNA. KITS WILL BE PRODUCED THAT ENABLE THE SIMULTANEOUS SEQUENCING OF BOTH STRANDS OF MULTIPLE DNA FRAGMENTS USING AS LITTLE AS 20 FEMTOMOLES TEMPLATE. CAS IS BASED ON A STRATEGY INVENTED AT YALE UNIVERSITY AND DATA TO DATE HAVE SHOWN THAT: A) CAS IS FEASIBLE, B) POTENTIAL PROBLEM AREAS HAVE BEEN DEFINED, C) THERE IS A NEED TO DEFINE THE ROBUSTNESS OF CAS BY SEQUENCING ADDITIONAL DNA FRAGMENTS, AND D) CAS IS LIKELY ADAPTABLE TO THE SIMULTANEOUS SEQUENCING OF MULTIPLE TEMPLATES. COLLABORATIVE STUDIES BY BIOS AND YALE WILL BE DIRECTED AT: A) FINDING A THERMOSTABLE DNA POLYMERASE MOST COMPATIBLE WITH CAS, B) DETERMINING OPTIMAL CONDITIONS FOR SEQUENCING DNA OF DIFFERENT FRAGMENT LENGTHS, C) DEMONSTRATING ROBUSTNESS BY APPLYING CAS TO DIFFERENT TEMPLATES, AND D) DETERMINING AND IMPLEMENTING CONDITIONS FOR MULTIPLEX CAS. RESULTS OF THESE STUDIES WILL SHOW THAT DNA SEQUENCING CAN BE MADE SIMPLE, ACCESSIBLE TO MANY RESEARCHERS, AND PREFERABLE TO THE CURRENT INDIRECT METHODS FOR DETERMINING SEQUENCE VARIATION. MOREOVER, CAS WILL IMPACT LARGE SCALE SEQUENCING EFFORTS BY PROVIDING A METHOD FOR DIRECT SEQUENCING OF COSMID INSERTS IN A SINGLE ELECTROPHORETIC RUN.