NONISOTOPIC DETECTION OF NUCLEIC ACID HYBRIDIZATION

ADVANCES IN MOLECULAR BIOLOGY HAVE FOSTERED A NEW INVESTIGATIONAL AREA. THE ANALYSIS OF CELLULAR DNA FOR ONCOGENIC SEGMENTS, LATENT VIRUSES, POLYMORPHISMS, GENE AMPLIFICATION, AND REARRANGEMENTS HAS PERMITTED RESEARCHERS TO ESTABLISH MEANINGFUL RELATIONSHIPS BETWEEN EVENTS AT THE DNA AND RNA LEVEL AND VARIOUS DISEASE STATES. THE MOST SENSITIVE METHOD FOR THE DETECTION OF THESE EVENTS IS USING RADIOLABELED NUCLEIC ACID PROBES COUPLED WITH AUTORADIOGRAPHY. IF DNA ANALYSIS IS TO BECOME ROUTINELY USED IN THE CLINICAL LABORATORY, SENSITIVE NONRADIOACTIVE DETECTION METHODS MUST BE DEVELOPED. IN THIS RESEARCH, IT IS PLANNED TO DEVELOP A NOVEL PROCEDUREFOR THE DETECTION OF HYBRIDIZED NUCLEIC ACID PROBES. THIS PROCEDURE INCLUDES SEVERAL STEPS: (1) SOUTHERN OR NORTHERN HYBRIDIZATION PROCEDURES USING PROBES DERIVATIZED WITH ALKYL HALIDE GROUPS; (2) COVALENT ATTACHMENT OF DEXTROSE POLYMERS CARRYING MULTIPLE ZINC-EDTA COMPLEXES TO THE ALKYL HALIDE GROUPS OF THE MODIFIED PROBES; AND (3) THE RELEASE OF ZINC IONS TO REACTIVATE INACTIVE ZINC-FREE ALKALINE PHOSPHATASE CONTAINED IN AGAROSE GELS THAT ARE PLACED OVER THE MEMBRANE. SUBSEQUENTLY, COLOR WILL BE DEVELOPED IN THE AGAROSE GELS RATHER THAN ON THE MEMBRANE.