Detection of gene amplification by genomic hybridization to cDNA microarrays.

Gene amplification is one of the major mechanisms of oncogene activation in tumorigenesis. To facilitate the identification of genes mapping to amplified regions, we have used a technique based on the hybridization of total genomic DNA to cDNA microarrays. To aid detection of the weak signals generated in this complex hybridization, we have used a tyramide-based technique that allows amplification of a fluorescent signal up to 1000-fold. Dilution experiment suggests that amplifications of 5-fold and higher can be detected by this approach. The technique was validated using cancer cell lines with several known gene amplifications, such as those affecting MYC, MYCN, ERBB2, and CDK4. In addition to the detection of the known amplifications, we identified a novel amplified gene, ZNF133, in the neuroblastoma cell line NGP. Hybridization of NGP cDNA on an identical array also revealed over expression of ZNF133. Parallel analysis of genomic DNA for copy number and cDNA for expression now provides rapid approach to the identification of amplified genes and chromosomal regions in tumor cells.

Fluorochrome-labeled tyramides: use in immunocytochemistry and fluorescence in situ hybridization.

The peroxidase-mediated deposition of hapten- and fluorochrome-labeled tyramides has recently been shown to increase the sensitivity of immunofluorescence and fluorescence in situ hybridization techniques. We have evaluated a number of red, green, and blue fluorescent tyramides for detection of antigens in tissue sections and cytospin preparations and for the detection of hapten- and horseradish peroxidase-labeled probes hybridized in situ to cells and chromosomes. With few exceptions, all fluorescent tyramide-based methods provided a considerable increase in sensitivity compared to conventional immunofluorescence and FISH methods.

Detection of HIV-1 DNA in crude cell lysates of peripheral blood mononuclear cells by the polymerase chain reaction and nonradioactive oligonucleotide probes.

The aim of this study was to detect HIV-1 proviral DNA in lysates of peripheral blood mononuclear cells (PBMCs) by the polymerase chain reaction (PCR) and hybridization with a nonradioactive probe. PBMCs were lysed in 1% Triton X-100. PCR was then carried out using primers complementary to a conserved region of the HIV-1 pol gene. Bracket and nested amplification protocols were used. Products were identified by dot-blot hybridization or agarose gel electrophoresis and Southern hybridization, using an alkaline phosphatase-linked oligonucleotide probe specific for amplified sequences. Colorimetric and chemiluminescent substrates were used. HIV-1 DNA was detected in PBMCs of 57/59 HIV-1-seropositive individuals, 8 of which were positive only following the use of nested primers. Of 12 seropositive samples that were negative by other HIV-1 diagnostic tests (PBMC coculture and serum p24 antigen detection), 11 were positive by PCR. PCR using PBMC lysates is a very sensitive method of detecting HIV-1 proviral sequences. The use of nested primers appears to increase the sensitivity of the procedure.