Development and application of “Catching-whole-mutations-in-genome method" that aims at health promotion activity
| Project/Area Number |
17500496
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied health science
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| Research Institution | National Cardiovascular Center Research Institute |
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Principal Investigator |
TAKAGI Atsuko National Cardiovascular Center Research Institute, 薬理部, Senior staff (90179416)
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| Co-Investigator(Kenkyū-buntansha) |
IKEDA Yasuyuki National Cardiovascular Center Research Institute, 病因部, Senior staff (90176107)
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| Project Period (FY) |
2005 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥3,850,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥450,000)
Fiscal Year 2007: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
Fiscal Year 2006: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2005: ¥900,000 (Direct Cost: ¥900,000)
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| Keywords | mutation / heterozygote / lipoprotein lipase / carbodiimide / heteroduplex / MutS / hyperlipidemia / hypertriglyceridemia / メタボリックシンドローム |
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| Research Abstract |
【Purpose】 Our aim is to develop a new method (“Catching-whole-mutations-in-genome method"; CWHG method) that can detect mutations of genes related to known and unknown diseases. 【Method】 Detection of heteroduplexs formed after heat treatment and slow cooling was carried out with biotinylated carbodiimide or mutation recognition protein MutS. Lipoprotein lipase (LPL) gene related to hypertriglyceridemia was used as a model. 【Result 1: Efficiency of double strand DNA (dsDNA)formation】 DNA fragment was treated with slow cooling after 5 min at 95℃. Efficiency of dsDNA formation was examined with nondenaturing gel. Most of the samples were detected as dsDNA in case of more than concentration of 0.1pmol DNA/ul. 【Result 2: Efficiency of heteroduplex formation】 Insertion mutations of four and eight bases were used to facilitate detection of heteroduplex. Efficiency of heteroduplex formation was 40%. This result was satisfactory, because maximal efficiency of heteroduplex was 50%. 【Result 3: Re
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activity between heterodupex and carbodiimide, and effect of reinforcing reagent of matching DNA region】 Specificity of reactivity between heteroduples and carbodiimide was low. Although reinforcing reagent of matching DNA region was used, the specificity was not improved. 【Result 4: Reactivity between heterodupex and MutS, and effect of reinforcing reagent of matching DNA region】 Specificity of reactivity between heteroduples and MutS was high, but all types of heteroduplex were not reacted. Reinforcing reagent of matching DNA region didn't improve this point. The G105R mutation, which was not detected at any SSCP conditions employed, was detected by reaction between heteroduplex and MutS. 【Result 5: Newly identified mutation in LPL gene】 The accumulation of new mutations is important for the establishment of CWHG method and early diagnosis of hypertriglyceridemia. Compound heterozygosity of a novel large deletion (54kb) from5' upstream region to intron 1 and Y61X in LPL gene was identified in a hypertriglyceridemic Japanese patient. Less
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Report
(4 results)
Research Products
(50 results)
