The applicants propose development of an efficient hybridization screening test for mutations in the lacI gene based on a microwell transfer pin approach and a """"""""probe"""""""" pooling strategy. The goal is to provide """"""""mutational fingerprints or spectra"""""""" capable of defining levels and mechanism of mutation induced by individual mutagenic compounds. The test will use Stratagene's BigBlue transgenic system to recover mutants. However, the present proposal seeks to develop a strategy whereby fluorescein- labeled oligonucleotides probes complementary to selected lacI mutations are specifically annealed to biotin-labeled PCR products immobilized on streptavidin-coated pins. Positive hybridization events are revealed by a colorimetric reaction using anti-fluorescein-horse radish peroxidase conjugates in microwells.
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