2000 Fiscal Year Final Research Report Summary
APPLICATION OF GENE TIP MICRO ARRAY FOR GENETIC DIAGNOSIS
| Project/Area Number |
11557201
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| Research Category |
Grant-in-Aid for Scientific Research (B).
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
Human genetics
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| Research Institution | TOHOKU UNIVERSITY |
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Principal Investigator |
SUZUKI Yoichi TOHOKU UNIV, MEDICAL GENETICS, ASSOCIATE RESEARCHER, 大学院・医学系研究科, 助手 (80216457)
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| Co-Investigator(Kenkyū-buntansha) |
NARISAWA Kuniaki TOHOKU BUNKA GAKUEN UNIV, SCI & WELFARE PROFESSOR, 医療福祉学部, 教授 (90004647)
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| Project Period (FY) |
1999 – 2000
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| Keywords | DNA micro array / inborn error of metabolism / biotin / holocarboxylase synthetase / muscular dystrophy / genetics diagnosis |
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| Research Abstract |
INTRODUCTION Duchenne muscular dystrophy (DMD) is caused by a defect of dystrophin gene. Seventy percent of patients have large deletion of the dystrophin gene, of which involvement of multiple exons are common. Several types of multiplex PCR methods were developed and used in clinical practice. However, the detection efficiency of the methods is not satisfactory and complexities of the procedure need to be improved. To develop more efficient genetic diagnosis method of this disease, we used DNA micro array technology.
METHODS DNA micro array was constructed using GTMASS system, a product of Nippon Laser Company. All coding exons were amplified with PCR and purified with ethanol precipitation. The DNA fragments were spotted on the poly-lysine coated slide glasses with the GTMASS spotting machine. cDNA was synthesized from total RNA from control and patient lymphoblast cell lines. The dystrophin mRNA coding sequence was amplified with PCR as 10 overlapping fragments which was Cy5' labele
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d during the PCR cycles. Cy5'dUTP was included in the PCR reaction mixture. Hybridization was performed with Cy5 labeled cDNA and DNA spotted slide glass in the 6XSSC.The slide glasses were washed with 6XSSC at 50℃. Signals of hybridized probes were detected with slide scanner of GTMASS system.
RESULTS DNA micro array that contains exons 43 to 51 successfully detected the exon 44 deletion in the patient DNA sample whose defect was established with multiplex PCR method previously, suggesting that this method are useful for detection of exon deletion of the patients with DMD.Fixation of oligonucleotide that is less than 50 bases in length was not enough to detect hybridized signals. Thus, it was difficult to perform allele specific oligonucleotide hybridization or primer extension method.
CONCLUSION DNA micro array technique was successfully applied for the detection of a large deletion of the gene. The disease such as DMD is a good target disease to this method. To detect a single base change, DNA micro array is not a good choice at this moment. However, by improving the efficiency of fixation of oligonucleotides on the glass surface, the method will potentially a replacement of other current methods. Less
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[Publications] Aoki, Y., Li, X., Sakamoto, O., Hiratsuka, M., Akaishi, H., Xue, L., Briones, P.Suormala, T., Baumgartner, R., Suzuki, Y., and Narisawa, K.: "Identification and characterization of mutations in patients with holocarboxylase synthetase deficiency."Human Genetics. 104. 143-148 (1999)
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「研究成果報告書概要(欧文)」より
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[Publications] Hou, D., Kure, S., Suzuki, Y., Hasegawa, Y., Hara, Y., Inoue, T., Kida, Y., Matsubara, Y., Narisawa, K.: "Glycogen storage disease type Ib : structural and mutation analysis of the microsomal glucose-6-phosphate transporter gene."Am.J.Med.Genet. 86. 254-257 (1999)
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「研究成果報告書概要(欧文)」より
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[Publications] Sakamoto, O., Suzuki, Y., Aoki, Y., Li, X., Hiratsuka, M., Suormala, T, Baumgartner, R., Gibson, M., and Narisawa, K.: "Relationship between kinetic properties of mutant enzymes and biochemical and clinical responsiveness to biotin in holocarboxylase synthetase deficiency."Ped.Res.. 46. 671-676 (1999)
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「研究成果報告書概要(欧文)」より
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[Publications] Sakamoto, O., Suzuki, Y., Li, X., Aoki, Y., Hiratsuka, M., Holme, E., Kudoh, J., Shimizu, N., and Narisawa, K.: "Diagnosis and mutation analysis of an atypical case of holocarboxylase synthetase deficiency."Eur.J.Pediatr.. 159. 18-22 (2000)
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[Publications] Fujii K, Matsubara Y, Akanuma J, Takahashi K, Kure S, Suzuki Y, Imaizumi M, linuma K, Sakatsume O, Rinaldo P, Narisawa K: "Mutation detection by TaqMan-allele specific amplification : Application to molecular diagnosis of glycogen storage disease type la and medium-chain acyl-CoA dehydrogenase deficiency"Hum.Mutat.. 15. 189-196 (2000)
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「研究成果報告書概要(欧文)」より
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[Publications] Akanuma, J., Nishigaki, T.Fujii, K., Matsubara, Y., Inui, K., Takahashi, K., Kure, S., Suzuki, Y., Ohura, T., Miyabayashi, S., Ogawa, E., linuma, K., Okada, S., Narisawa, K.: "Glycogen storage disease type la : molecular diagnosis of 51 Japanese patients and characterization of splicing mutations by analysis of ectopically transcribed mRNA from lymphoblastoid cell."J.Med.Genet.. 91. 107-112 (2000)
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「研究成果報告書概要(欧文)」より
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[Publications] Okubo, M., Horinishi, A., Suzuki, Y., Murase, T., Hayasaka, K.: "Compound Heterozygous Patient With Glycogen Storage Disease Type III : Identification of Two Novel AGL Mutations, a Donor Splice Site Mutation of Chinese Origin and a 1-bp Deletion of Japanese Origin."Am.J.Med.Genet.. 93. 211-214 (2000)
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「研究成果報告書概要(欧文)」より
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[Publications] Takahashi, K., Akanuma, J., Matsubara, Y., Fujii, K., Kure, S., Suzuki, Y., Wataya, K., Sakamoto, O., Aoki, Y., Ogasawara, M., Ohura, T., Miyabayashi, S., Narisawa, K.: "Heterogeneous mutation in the glucose-6-phospatase gene in Japanese patients with glycogen storage disease type 1a."Am.J.Med.Genet.. 92. 90-94 (2000)
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「研究成果報告書概要(欧文)」より
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[Publications] Suzuki, Y., Lanner, C., Chen Bi, Hong Zhang, Cooper, L.D., Melissa M., Bowker-Kinley, DePaoli-Roach, A.: "A.Gene structure and expression of the targeting subunit, RGL, of the muscle-specific glycogen-associated type 1 protein phosphatase, PP1G."Archiv.Biochem.Biophys.. (in press). (2001)
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「研究成果報告書概要(欧文)」より
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[Publications] Suzuki, Y., Lanner, C., Kim, J.-H., Vilardo, P.G., Zhang, H., Yang, J., Cooper, L., Steele, M., Kennedy, A., Scrimgeour, A.Lawrence Jr., J., C., DePaoli-Roach, A.A.: "Insulin control of glycogen metabolism in knockout mice lacking the muscle-specific protein phosphatase PP1G/RGL."Mol.Cell.Biol.. (in press). (2001)
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