| 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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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥4,700,000 (Direct Cost: ¥4,700,000)
Fiscal Year 2000: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1999: ¥2,600,000 (Direct Cost: ¥2,600,000)
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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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