An in situ DNA hybridization assay suitable for detecting target nucleic acid in single intact cells by automatic flow analysis 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 standards 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 DNA probes 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 route DNA hybridization technology to the chemical laboratory.
Thesaurus Terms: chemical synthesis, design and production, nucleic acids, diagnostic tests, design, development and evaluation of diagnostic tests, nucleic acids, nucleic acid probes, biotechnology, cell sorting, nucleic acid hybridization biological transport, membrane permeability and transport, diagnostic tests, cytodiagnosis, dyes, fluorescent dyes and probes, immunopathology, immunologic deficiency disorders, acquireddeficiency disorders, aids diagnosis, nucleic acids structure, nucleosides (tides) sequence, nucleotides, oligonucleotides, peroxidases, phosphomonoesterases, alkaline phosphatase, physical properties, solutions, virus diseases, viruses, herpesviridae, alphaherpesvirinae, viruses, herpesviridae, betaherpesvirinae, viruses, retroviridae, human immunodeficiency viruses (hiv) nucleic acid hybridization, in situ hybridization, tissue (cell) culture
