International Mammalian Genome Society

The 16th International Mouse Genome Conference (2002)


POSTER 110 - ENU MOUSE MUTAGENESIS PROJECT IN RIKEN GSC: HIGH-THROUGHPUT DETECTION OF POINT MUTATIONS

H Sezutsu
Population and Quantitative Genomics Team, RIKEN Genomic Sciences Center

1) Sakuraba Y, 1) Takahasi KR, 2) Wakana S, 2) Noda T, 2) Shiroishi T, 1) Gondo Y
1) Population and Quantitative Genomics Team and 2) Mouse Functional Genomics Research Group, RIKEN Genomic Sciences Center

For gene-driven mutagenesis as well as candidate gene approach, it is necessary to develop an efficient system for detection of the sites of novel point mutation in the mouse genome. It must be high-throughput, cost effective and accurate. For this purpose we designed more than 200 pairs of primers to amplify target sequences of C57BL/6 genome. The size of these amplicons ranges from 200 bp to 1.2 kb. We have been testing the efficacy of the point mutation detection in the amplicons by using direct sequencing, TGCE and DHPLC.  We have firstly tried the direct sequencing method to identify point mutations in target genes. The sequencing data have been fairly well produced. However the method is relatively expensive and slow, and the analyses of numerous chromatogram data are laborious and error-prone. It is thus necessary to develop some primary screening method with high-resolution and high-throughput system to detect the site of mutations. We have adopted the TGCE (Temperature Gradient Capillary Electrophoresis) method, which can effectively detect heteroduplex DNA. It is relatively low cost and high-throughput. We have been trying to optimize temperature gradient and ramp time for the detection of heteroduplex DNA in each amplicon, considering the GC contents and the melting temperature point. In some amplicons, we have found the optimal parameter for screening of point mutations. We have also tried DHPLC (Denaturing High Performance Liquid Chromatography) method for detecting the heteroduplex DNA. We will discuss about the efficiency of these methods for high-throughput detection of point mutations.


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