| Project/Area Number |
12557235
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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 |
Laboratory medicine
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| Research Institution | HOKKAIDO UNIVERSITY |
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Principal Investigator |
KINJO Masataka Hokkaido Univ. Res. Inst. Elect. Sci. Asso. Prof., 電子科学研究所, 助教授 (70177971)
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| Co-Investigator(Kenkyū-buntansha) |
ISHIDATE Fumiyoshi Carl Zeiss Co. Ltd. Div. Microscopy. Division Manager, 顕微鏡システム研究グループ, グループ長
NOMURA Yasutomo Yamagata University, Dept. Bio-System Eng. Asso. Prof., 工学部, 助教授 (80237883)
NISHIMURA Goro Hokkaido Univ. Res. Inst. Elect. Sci. Inst., 電子科学研究所, 助手 (30218193)
HORI Kunio Novus Gene Inc. R&D Division, Manager, 研究開発部, 課長(研究職)
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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 |
¥9,700,000 (Direct Cost: ¥9,700,000)
Fiscal Year 2002: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2001: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 2000: ¥6,100,000 (Direct Cost: ¥6,100,000)
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| Keywords | FA-PCR / PCR / fluorescence measurement / fluorescence correlation spectroscopy / gene expression / single molecule detetion / SNP / RFLP |
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| Research Abstract |
PCR (polymerase chain reaction) and modified PCR method, such as RT-PCR (reverse transcriptase) have been used for detection of specific gene and genomic information. However, conventional PCR analysis contains many processes such as enzymatic reaction, product separation and determination.
Asymmetric PCR, in which the concentrations of primers are unequal, was introduced to make a single-strand DNA. We focused not on a single-stranded DNA (ssDNA) but on another product, a double-stranded one (dsDNA). When the concentration of the fluorescence labeled primer is lower than that of of the other primer in amplification, fluorescent dsDNA and non-fluorescent ssDNA can be expected as products of the reaction. Under ideal conditions, all of the fluorescence-labeled primer (low-concentration primer) is incorporated into dsDNA after the proper number of PCR cycles. From the viewpoint of fluorescence molecules, short ssDNA (fluorescent primer) is elongated into long dsDNA (fluorescent amplified DNA) during the FA-PCR (fluorescence-labeled primer based asymmetric PCR).
We determined the product of FA-PCR by FCS (Fluorescence Correlation Spectroscopy) under homogenous condition. FCS provides the average number of molecules in the volume element and the diffusion constant of the molecule.
In this project we determined the yield of several kind of DNA in FA-PCR using FCS as a simple tool for detection of specific genome and sequence.
We reported the sensitivity and simplicity of FCS in detecting dsDNA in FA-PCR without another extra specific probe. The results indicated the combination of FCS and FA-PCR could detect the target gene at the single copy level at the initial concentration in 25 micro litter solution without using any separation or purification method such as gel electrophoresis.
We also developed FA-PCR detection system based FCS. The system consisted with sample carrier stage and data evaluation computer system.
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