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

使用DNA微阵列开发肌肉疾病相关基因中SNP的搜索方法

• 批准号: 12470532
• 负责人: TSUKAHARA Toshifumi
• 金额: $ 9.09万
• 依托单位: National Institute of Neuroscience, NCNP
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12470532/
• 关键词: cDNA microarry; SNPs; database; fluorescence-labeled; misannealing; PCR; cloning; high-fidelity cDNA; cDNAマクロアレイ; _cDNAマイクロアレイ; SNP_s; 単塩基多型; データーベース

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

为了建立方便的搜索方法，该方法使用cDNA微阵列识别单个核苷酸多态性SNP，我们开发了具有高保真性cDNA的大型微阵列。我们为在骨骼或心脏肌肉中表达的虚拟cDNA构建了一个高度冗余的人类单例数据库。数据库中的每个基因片段用特定的引物，肌肉cDNA池和高保真KOD+ DNA聚合酶进行扩增，然后通过测序克隆并确认。最后，由高保真cDNA克隆产生了具有5,760个斑点的大型cDNA微阵列。首先，我们确认了微阵列的灵敏度和可重复性。我们采用泰米酰胺信号扩增系统进行标记和检测。在使用人骨骼肌总RNA作为靶标的一式三份实验中，我们的微阵列在100-100000相对荧光强度的范围内显示出良好的可重复性（r = 0.94-0.98）。每个目标点的荧光强度是线性的……在L到4μg的总RNA范围内增加了更多。因此，1μg的总RNA足以分析，我们的微阵列表现出较低的背景和良好的分辨率。Bortoluzzi等。并显示了转录曲线数据库中人类骨骼肌的EST克隆相对丰度和Okubo等人的相对丰度。还显示了人体图数据库中每个基因的基因表达排名。我们的微阵列的结果与这些数据并不矛盾。并且，在同一基因的多个问题中，在每个目标点上获得了相似的强度。用于检测SNP，尝试直接荧光标记为突变位点。但是，背景是如此之高，因为将荧光掺入了基因片段的两端。接下来，我们试图建立DNA修复酶错误退火位置的检测方法，因为这种蛋白可以检测突变。我们克隆了这种基因和合成的重组蛋白。但是，不可能分离可以在体外活性的重组蛋白。我们需要继续进一步的努力来隔离功能蛋白。较少的

项目成果

Yeon-Jeong Kim, Satoru Noguchi, Yukiko K. Hayashi, Toshifumi Tsukahara, Takao Shimizu, Kiichi Arahata: "The product of an oculopharyngeal muscular dystrophyu gene, poly(A)-binding protein 2, interacts with SKIP and stimulates muscle-specific gene expressi

Yeon-Jeong Kim、Satoru Noguchi、Yukiko K. Hayashi、Toshifumi Tsukahara、Takao Shimizu、Kiichi Arahata：“眼咽肌营养不良症基因的产物 Poly(A) 结合蛋白 2 与 SKIP 相互作用并刺激肌肉特异性基因

塚原俊文,荒畑喜一: "筋ジストロフィー"原因遺伝子と病態の関連性""遺伝子医学Medical Do. 15号. 83-88 (2001)

Toshifumi Tsukahara、Kiichi Arahata：“肌肉萎缩症”致病基因与病理学之间的关系”Genetic Medicine Medical Do. 15. 83-88 (2001)

W Aerbajinai, T Ishihara, K Arahata, T Tsukahara: "Increased expression level of the splicing variant of SIP1 in motor neuron diseases"Int. J. Biochem. Cell Biol.. 34・6. 699-707 (2002)

W Aerbajinai、T Ishihara、K Arahata、T Tsukahara：“运动神经元疾病中 SIP1 剪接变体的表达水平增加”Int. Biochem. 34・6 (2002)。

T Tsukahara, K Arahata: "Methods in Molecular Medicine-Neurogenetics : Methods and Protocols Nicholas T Potter, Ed"Humana Press(印刷中). (2002)

T Tsukahara、K Arahata：“分子医学-神经遗传学方法：方法和方案 Nicholas T Potter，Ed”Humana Press（出版中）。

YJ Kim, S Noguchi, YK Hayashi, T Tsukahara 他: "The product of an oculopharyngeal muscular dystrophy gene, poly(A)-binding protein 2, interacts with SKIP and stimulates muscle-specific gene expression"Hum Mol Genet.. 10. 1129-1139 (2001)

YJ Kim、S Noguchi、YK Hayashi、T Tsukahara 等人：“眼咽肌营养不良症基因的产物，多聚 (A) 结合蛋白 2，与 SKIP 相互作用并刺激肌肉特异性基因表达”Hum Mol Genet.. 10. 1129-1139 (2001)