| Budget Amount *help |
¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 2006: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2005: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Research Abstract |
LICOR mutation detection system was introduced to find mutations among 5720 ENU-treated cryopreserved medakafish. The system takes advantage of mismatch-recognizing nuclease, which would generate a shorter fragment from heteroduplex formed between wild type and mutated alleles. Since it turned out that this method is time-and labor-consuming, and a considerable number of false positives and false negatives were found, the 'direct sequencing' method was tested. Here, all the PCR products were directly sequenced, assembled by Phred/Phrap, and the heterozygous sites were evaluated by polyphred. Applying this method to p53 locus, we have achieved the satisfactory mutation recovery rate albeit the running cost was by far high compared to TILLING.
The amplicon was set for exons 3-6 of medaka Chk1, which encode the conserved kinase domain. 1920 mutagenized medaka were screened for mutations by using direct sequencing method. 3 single nucleotide polymorphisms and 9 ENU-induced mutations were recovered. The latter entity consisted of 6 intronic, 2 silent and 1 missense (His64Gln) mutations. Unfortunately, there were no mutations that would disrupt the functions of Chk1 kinase, such as nonsense mutation, splice site mutation, or the mutation that would introduce the amino acid change in the catalytic active site. This low rate of missense mutation recovery could be in part because of the rather short coding region in the selected amplicon (433 bp out of 849 bp screened).
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