Development of the searching method for SNPs in responsible genes for muscular diseases using the DNA microarray

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To establish the conveniently searching method which identifies single nucleotide polymorphisms, SNPs, using a cDNA microarray, we developed the large-scale microarray with high-fidelity cDNAs. We constructed a highly nonredundant human singleton database for virtual cDNAs expressed in skeletal or cardiac muscle. Each fragment of genes in the database was amplified with specific primers, muscle cDNA pools and high-fidelity KOD+ DNA polymerase, then cloned and confirmed by sequencing. Finally, the large-scale cDNA microarray with 5,760 spots was produced from high-fidelity cDNA clones.First, we confirmed sensitivity and reproducibility of our microarray. We employed tyramide signal amplification system for labeling and detection. In triplicate experiments using human skeletal muscle total RNA as a target, our microarray showed good reproducibility (R=0.94〜0.98) in the range of the 100〜100000 relative fluorescence intensity. And the fluorescence intensity of each target spot was linearly … More increased in the range of l〜4μg of total RNA. Therefore, oneμg of total RNA was enough to analyze and our microarray showed low background and good resolution.Bortoluzzi et al. and showed relative abundance of EST clones from human skeletal muscle in the Transcriptional Profile database and Okubo et al. also showed gene expression ranking of each gene in the BodyMap database. The results from our microarray were not contradictory to those data. And, the similar intensity was obtained on each target spot in multiple probes of the same the gene.For detection of SNPs, the direct fluorescent-labeling to the mutated-site was tried. However, the background was so high, since the fluorescence were incorporated in both ends of the gene fragments. Next, we tried to establish the detection method for the mis- annealing position by DNA repair enzymes because such proteins can detect mutation. We cloned such genes and synthesized recombinant proteins. However, it was not possible to isolate recombinant proteins which can be active in vitro. We need to continue further efforts to isolate functional protein. Less