Host: The Japan Radiation Research Society
Mutation of Sfpi1 (PU.1) is crucial in the development of myeloid leukemia in mice exposed to radiation. The mutation is mostly missense point mutations at codon 235 in the DNA-binding Ets domain of PU.1. While they are unlikely to be a direct consequence of radiation-induced DNA damage, it is not evident when and how often this type of mutation arises in hematopoietic cells. To facilitate experimental approach to this issue, we have developed a technique that is able to detect a small number of mutated cells in the vast majority of non-mutated cells. The technique is based on the wild-type blocking PCR, in which LNA (locked nucleic acid) oligonucleotide blocks amplification of wild-type DNA during PCR while permitting amplification of mutants. A primer set to amplify a 443bp region within exon 5 of Sfpi1 was applied simultaneously with an 11mer LNA oligonucleotide that covers the mutation hot-spot in leukemic cells. Adjusting reaction condition has enabled two-step PCR to detect mutant DNA in 103 times amount of wild-type DNA. DNA samples of exposed mice that are not affected with overt leukemia is being analyzed for the leukemia-specific Sfpi1 mutation with this technique.
