CHEMICAL METHODS FOR MAKING NON-RADIOACTIVE DNA PROBES

NONRADIOACTIVE DNA-BASED DETECTION SYSTEMS OFFER AN ATTRACTIVE NEW APPROACH TO THE CLINICAL DIAGNOSIS OF A VARIETY OF VIRAL AND BACTERIAL PATHOGENS. CURRENT METHODS FOR LABELING DNA BY ENZYMATIC PROCEDURES ARE TIME CONSUMING,LABOR INTENSIVE, AND NOT WELL SUITED FOR LARGE-SCALE PRODUCTION. THIS PROPOSAL DESCRIBES A NOVEL METHOD FOR THE CHEMICAL, NONRADIOACTIVE LABELING OF DNA PROBES WHICH OVER- COMES THE PROBLEMS INHERENT IN ENZYMATIC PROCEDURES. THIS SIMPLE, TWO-STEP PROCEDURE INVOLVES THE ATTACHMENT OF A HYDRAZIDE LINKER ARM TO CYTOSINE RESIDUES, FOLLOWED BY COUPLING OF A REPORTER MOLECULE TO THE LINKER ARM. IN PHASEI, OPTIMAL CONDITIONS WILL BE DETERMINED FOR LINKER ARM COUPLING AND FOR ATTACHMENT OF SEVERAL REPORTER MOLECULES (BIOTIN, TEXAS RED, FLUORESCEIN) TO THE MONONUCLEOTIDE 2'- DEOXYCYTIDYLIC ACID. THE MOST SUITABLE PROCEDURES WILL BE EMPLOYED IN THE LABELING OF SINGLE AND DOUBLE-STRANDED M13 DNA. THE HYBRIDIZABILITY AND DETECTION SENSITIVITY OF THESENEWLY CONSTRUCTED PROBES WILL BE BRIEFLY EXAMINED. THE LABELING OF OTHER DNAS AND MORE DETAILED STUDIES ON HYBRID- IZATION AND SENSITIVITY MAY BE CARRIED OUT IN PHASE II. THECHEMICAL LABELING PROCEDURE OUTLINED HERE SHOULD REPRESENT A MAJOR ADVANCE IN THE COMMERCILIZATION OF DNA-BASED TECHNOLOGY.