| Project/Area Number |
12555237
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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 |
工業分析化学
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| Research Institution | RIKEN (2002)
Kyushu University (2000-2001) |
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Principal Investigator |
MAEDA Mizuo RIKEN, Bioengineering Laboratory, Chief Scientist, バイオ工学研究室, 主任研究員 (10165657)
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| Co-Investigator(Kenkyū-buntansha) |
TAKARADA Tohru Graduate School of Engineering, Department of Applied Chemistry, Instructor, 大学院・工学研究院, 助手 (30336010)
村田 正治 九州大学, 大学院・工学研究院, 助手 (30304744)
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| Project Period (FY) |
2000 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥9,500,000 (Direct Cost: ¥9,500,000)
Fiscal Year 2002: ¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 2001: ¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 2000: ¥4,800,000 (Direct Cost: ¥4,800,000)
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| Keywords | DNA / Capillary Electrophoresis / Polyacrylamide / Gene Diagnosis / Single Nucleotide Mutation / SNP / Separation Analysis / Oncogene / 電気泳動 / 高分子 / 遺伝子 |
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| Research Abstract |
Gene mutations have been studied in relation to genetic diseases and cancers, and the detection of DNA single-base mutations should be helpful in making medical diagnoses of these disorders. A number of methods such as single-strand conformation polymorphism (SSCP) and restriction fragment length polymorphism (RFLP) have been developed for detecting point mutations and single nucleotide polymorphisms (SNPs), but they are still time-consuming and laborious. Rapid, facile and sensitive detection methods are required.
In this study, we have developed an affinity capillary electrophoresis for a single-base difference detection of oligodeoxyribonucleotides using a DNA-polyacrylamide conjugate. The DNA-polyacrylamide conjugate was synthesized through copolymerization between acrylamide and ODN macromonomer, which was obtained by coupling reaction between methacryloyloxysuccinimide and ODN having amino-hexyl linker at its 5'-end. The DNA-polyacrylamide conjugates injected into a capillary can serve as pseudo-immobilized affinity ligands because they behave similarly to non-ionic polyacrylamide in terms of migration rate. This novel affinity capillary electrophoresis was successfully applied to separation of oligodeoxyribonucleotides (60 mer) having an oncogene (K-ras) sequence and a version with single-base substituted within 30 minutes. Moreover, sample DNAs obtained from human cells through PCR amplification can be subjected to this separation system.
In conclusion, by using DNA-polyAAm conjugate as an affinity ligand we can separate DNAs having a single-base difference on capillary electrophoresis system. This analytical system is promising for the novel gene mutation assay.
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