SOLUTION IN-SITU HYBRIDIZATION FOR FLOW ANALYSIS

AN IN SITU DNA HYBRIDIZATION ASSAY SUITABLE FOR DETECTING TARGET NUCLEIC ACID IN SINGLE INTACT CELLS BY AUTOMATIC FLOWANALYSIS WILL BE INVESTIGATED. PROTOCOLS FOR FIXING CELLS IN SUSPENSION AND DNA DENATURATION WILL BE DEVELOPED. EMPHASIS WILL BE PLACED ON MINIMIZING CELL CLUMPING FOR COMPATIBILITY WITH FLOW CYTOMETRY AND CREATING CONDITIONS FOR OPTIMUM EXPOSURE OF THE NUCLEIC ACIDS. THE TARGET SEQUENCE WILL BE DETECTED BY LOW MOLECULAR WEIGHT SYNTHETIC OLIGONUCLEOTIDE PROBES DIRECTLY LABELED WITH ENZYMES OR FLUOROPHORES. THESE PROBES HAVE BEEN SHOWN TO BE HIGHLY SENSITIVE, WITH UNALTERED SPECIFICITY AND KINETICS AND EXHIBIT REMARKABLY LOW BACKGROUND BINDING IN STANDARD IN SITU AND FILTER-BASED FORMATS. ENHANCED CELL PERMEABILITY, BY ELECTRONIC OR CHEMICAL MEANS WILL BE EXPLORED, IF NECESSARY, TO MAXIMIZE PROBE PENETRATION AND SUBSEQUENT ELIMINATION OF UNBOUND MATERIAL. A SYSTEM CONSISTING OF DNAPROBES SPECIFIC FOR HERPES SIMPLEX VIRUS-INFECTED TISSUE CULTURE CELLS WILL SERVE AS A MODEL. THIS WILL BE FOLLOWED BY SYSTEMS FOR CYTOMEGALOVIRUS AND HUMAN IMMUNODEFICIENCY VIRUS. THE NATURAL SENSITIVITY OF A DIRECTLY DETECTED IN SITU HYBRIDIZATION FORMAT COUPLED WITH THE ABILITY TO AUTOMATICALLY SCAN THOUSANDS OF INDIVIDUAL CELLS PER MINUTE WILL PROVIDE THE POTENTIAL TO DELIVER ROUTINE DNA HYBRIDIZATION TECHNOLOGY TO THE CHEMICAL LABORATORY.