1996 Fiscal Year Final Research Report Summary
STUDY FOR MECHANISM OF TRIPLEX USING SINGLE MOLECULES DETECTION
| Project/Area Number |
07808072
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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 |
Biophysics
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| Research Institution | HOKKAIDO UNIVERSITY |
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Principal Investigator |
KINJO Masataka Hokkaido Univ.Res.Inst.Elect.Sci., Res.Assoc., 電子科学研究所, 助手 (70177971)
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| Co-Investigator(Kenkyū-buntansha) |
NISHIMURA Goro Hokkaido Univ.Res.Inst.Elect.Sci., Res.Assoc., 電子科学研究所, 助手 (30218193)
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| Project Period (FY) |
1995 – 1996
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| Keywords | DNA / TRIPLEX / FLUORESCENCE CORRELATION SPECTROSCOPY / FLUORESCENCE / AUTOCORRELATION / RHODAMIN / SINGLE MOLECULE / HYBRIDIZATION |
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| Research Abstract |
PURPOSE :
The formation process of triple helix DNA in an aqueous solution was analyzed using Fluorescence Correlation Spectroscopy in tiny volume at the single molecule detection. The triple helix DNA was made with fluorescence labeled oligo T15 as a probe DNA strand and homo purin : homo pyrimidine DNA as a target strand. Changing the translational diffusion time of probe DNA was used as indicator for the rate of producing of triplex.
We confirmed the FCS could be used as determination method for triplex in the first budget year, and we have applied the FCS method to analysis the formation of triplex in solution at the second budget year.
METHODS :
Various length of fluorescence labeled DNA (50bp to 7000bp) was synthesized using PCR method in the presence of fluorescence tagged dUTP (Rho-dUTP). The translational diffusion time of these DNA were measured with FCS and had been used as standard. In order to compare with the formation process of triplex, we measured the associate process of T15 to complementary strand DNA.
CONCLUSION :
The translational diffusion time of double strand DNA agreed with the theoretical values for a rod-shaped model. We could analyze the translational diffusion time of triplex DNA using same method with two components model since triplex has more rigid structure than double strand DNA.The results show the formation rate of triplex was slower than that of double helix. Also, about 80% of probe DNA bind to target double strand DNA while all of added probe DNA strand bind to target DNA in the case of double strand DNA.
The experiment with mismatch sequences in the target and/or the probe DNA will be analyzed in future.
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