The objective of Phase I is to develop simple, sensitive and unambiguous chemiluminescent detection of juxtaposed genes in chromosomal translocations associated with many cancers. We propose chemiluminescent assay formats based on simultaneous probing and detection of both genes of a translocation in a single Southern blot. Phase I will prove the feasibility by developing a Southern hybridization assay for the simultaneous detection of the juxtaposed bcl-2 oncogene and the JH DNA segment of the immunoglobulin heavy chain locus in the t(14;18) translocations. Simultaneous detection will be achieved using two types of chemiluminescent formats: a dual substrate that emits light only by the combined action of two different antibody-enzyme conjugates bound to the target DNA in close proximity to each other; and consecutive application of separate chemiluminescent substrates where the chemiluminescence generated from one probe in the first step is completely stopped in the second step when the second enzyme initiates light from the other probe. The proposed methods can be adapted to the detection of many different juxtaposed genes associated with cancers. The methods developed in Phase I will be used to Phase II to develop kits for rapid microtiter plate assays for gene rearrangements. PROPOSED COMMERCIAL APPLICATION: The proposed chemiluminescent assays should provide the basis for a new method and kits for detection of various gene arrangements resulting from chromosomal translocations associated with specific types of cancer. The proposed assays will be simpler and more rapid than current methods of detecting gene rearrangements and will permit unambiguous detection in a single test. The detection system can also be used in developing kits for developing minimal residual disease during cancer therapies.
Thesaurus Terms:biotechnology, gene rearrangement, luminescence, method development, oncogene, southern blotting alkaline phosphatase, bioassay, cardenolide, chromosome translocation, horseradish peroxidase, immunoglobulin gene polymerase chain reaction, tissue /cell cultureNational Cancer Institute (NCI)
