Method of detecting single gene copies in-situ
A method for detecting single copies of a gene in-situ using brightfield microscopy is used in detection of nucleic acid sequences. Probes are directly or indirectly labeled with alkaline phosphatase with NBT/BCIP used as the chromogen.
1. A method of visually detecting a single copy of the Her-2/neu gene in chromosomal DNA in an intact cell using brightfield microscopy, comprising:
heating the tissue or cell sample sufficiently to dissociate the native chromosomal target strands of Her-2/neu DNA;
contacting said tissue or cell sample with a digoxigenin-labeled nucleic acid Her-2/neu probe specific for the Her-2/neu gene under conditions that allow the re-hybridization of the labeled nucleic acid Her-2/neu probe and target strands of Her-2/neu DNA to form a target-probe duplex;
contacting the target-probe duplex with an anti-digoxigenin antibody under conditions allowing the antibody to bind to the label;
contacting the anti-digoxigenin antibody with an enzyme and a chromogen composition under conditions allowing the development of a visually detectable chromogen substrate signal at each target-probe duplex in the nucleus of the intact cell separate and distinct from the chromogenic signals of other copies of said chromosomal target nucleic acid sequence; and
detecting the chromogenic substrate signal visually using brightfield microscope conditions.
2. The method of claim 1 wherein the enzyme is selected from the group consisting of a phosphatase and a peroxidase.
3. The method of claim 1 wherein the chromogen is selected from the group consisting of NBT/BCIP, tetramethylbenzidine and diamino benzidine.