NON-RADIOACTIVE DETECTION SYSTEMS BASED ON ENZYME FRAGMENT COMPLEMENTATION

ASSAYS BASED ON DNA HYBRIDIZATION ARE VERY COMMONLY USED IN MOLECULAR BIOLOGY, CLINICAL DIAGNOSTICS, AND THE HUMAN GENOME PROJECT. HOWEVER, EFFICIENCY AND SENSITIVITY OF HYBRIDIZATINS ARE OFTEN LIMITED BY NON-SPECIFIC BINDING OF PROBE MOLECULES OR DETECTION SYSTEM COMPONENTS TO THE TARGET DNA OR MEMBRANE SUPPORTS. DETECTION METHODS FOR EXTREMELY SENSITIVE, RELIABLE, BACKGROUND-FREE DETECTION IN HYBRIDIZATION-BASED ASSAYS WILL BE DEVELOPED IN THIS PROJECT. THE SYSTEM WILL BE BASED ON TWO NEIGHBORING HYBRIDIZATION PROBES, EACH LABELED WITH ONE SUBUNIT OF ALKALINE PHOSPHATASE. INDIVIDUAL SUBUNIT ALKALINE PHOSPHATASE ARE INACTIVE, AND NON-SPECIFIC BINDING OF HYBRIDIZATION PROBES TO TARGET DNA OR MEMBRANE SUPPORT WILL THEREFORE NOT LEAD TO BACKGROUIND NOISE. UPON BINDING TO TWO PROBES NEXT TO EACH OTHER ON THE TARGET DNA, THE TWO ENZYME SUBUNITS CAN COMBINE AND FORM THE ACTIVE DIMERIC ENZYME WHICH CAN BE DETECTED WITH CHEMILUMINESCENT OR COLORIMETRIC SUBSTRATES. INCREASED HYBRIDIZATION SPECIFICITY RESULTS FROM THE NECESSITY OF TWO PROBES TO HYBRIDIZE NEXT TO ONE ANOTHER. IN PHASE I THE FEASIBILITY AND POTENTIAL OF SUCH BINARY DETECTION SYSTEMS WILL BE DEMONSTRATED. THE CONDITIONS FOR GENERATION, PURFICATION, STORAGE AND USE OF SUBUNIT-LABELED OLIGONUCLEOTIDE PROBES WILL BE DEFINED AND OPTIMIZED. FINALLY, THE BINARY DETECTION SYSTEMS WILL BE USED FOR CHEMILUMINESCENT MULTIPLEX SEQUENCING. THE EXTENSION OF PROTOCOLS WITH BINARY ENZYME-LABELED PROBES TO A LARGE VARIETY OF OTHER APPLICATIONS IN MOLECULAR BIOLOGY AND CLINICAL DIAGNOSTICS IS INTENDED TO TAKE PLACE IN PHASE II.