Technical improvement and development of automatic detection method for genomic mutation

Fluorescent in situ hybridization (FISH) method was improved to estimate the dose of radiation exposure. Cleavage of DNA molecules in lymphocyte was used as the detection parameter and the procedures for preparation of samples suitable for atomic force microscopy and laser microscopy were developed. When ordinal primers on the market were used for PCR, the products were generally too small (about 200 b.p.) for detection by FISH method. Therefore, dATP and biotin-labeled dUTP were linked to its 3'-end by treatment with TdT for 2 hours, resulting that the mean length of PCR products was ca. 1.5 Kb. After hybridization using this prove, signal amplification was carried out according to biotin-avidin detection method. Thus, fluorescent signals on chromosomes could be easily detected. When three primers, D6S105, D6S291 and D6S282 were used as primer, fluorescent signal was detectable at 3 sites on chromatin fiber. These results indicate that this method is available for the analysis of cleavage of chromosomes. However, the backgrounds were much varied depending on the way to wash the preparation after incubation with fluorescent particle to form biotin-avidin binding. Therefore, further improvement of this method was necessary to apply in practice. When chromosomes 13, 14 and 15 from lymphocytes exposed to X-ray were used as test samples, it was demonstrated that radio-sensitivity was variable depending on the contents of R band in each chromosome. (M.N.)