International Mammalian Genome Society

17th International Mouse Genome Conference

9-12 November 2003, Braunschweig, Germany


POSTER 56 - HIGH-THROUGHPUT MUTATION DISCOVERY BY MULTIPLEX TGCE (TEMPERATURE GRADIENT CAPILLARY ELECTROPHORESIS) IN MOUSE ENU-MUTAGENESIS

Sakuraba Y
Population and Quantitative Genomics Team, RIKEN GSC

Co-Authors: 1) Sezutsu H, 1) Takahasi KR, 1) Tsuchihashi K, 1) Kaneko S, 1) Fujimoto N, 1) Matsumoto R, 2) Ikeda A, 2) Karashima Y, 2) Inoue M, 2) Kaneda H, 2) Minowa O, 2) Wakana S, 2) Noda T, 2) Shiroishi T, 1) Gondo Y
Institutions: 1) Population and Quantitative Genomics Team, RIKEN GSC. 2) Mouse Functional Genomics Research Group, RIKEN GSC

Mutation discovery is one of the most important issues for the gene-driven ENU mutagenesis. For the high-throughput search of point mutations, we have adopted the Temperature Gradient Capillary Electrophoresis (TGCE) method. It is relatively low cost and high-throughput for mutation discovery. By using positive controls, we calibrated the running condition of TGCE for several target amplicons. We have so far screened 24 genes in 6000 G1 mice by TGCE and succeeded in finding novel 50 point-mutations in 19 genes. A few mutations were found to be identical probably due to the clonal expansion during the mutagenized spermatogenesis. Subtracting these duplicates, we concluded that there are 46 independent ENU-induced mutations by screening the total of 60 Mbp. Out of 24 genes screened, we have not been able to detect any mutations in 5 genes and have found only one mutation in the other 5 genes. The remaining 14 genes have been identified more than one mutation. The mutation rate of one in 1.3 Mb by TGCE seems to be lower than that obtained by the direct-sequencing method of one in approximately 1 Mb. In spite of somewhat lower detectability by using TGCE, the gene-driven approach concretely detect point mutations in the target genes. In most of the cases, multiple independent mutation alleles have been found so that it now becomes feasible and practical to study the functions of the genes from domain to domain separately. Recently we are screening about 8-9 Mb and finding 6 mutations on average per month.


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