| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2000: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1999: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Research Abstract |
The purpose of this research is to clarify the characteristic interaction between DNA strands in triple DNA helix by using fluorescence cross correlation spectroscopy (FCCS) method.
The use of autocorrelation analysis in fluorescence correlation spectroscopy (FCS) to monitor hybridization open new degree of sensitivity in the nucleic acid research, however the mass ratio of target strand must be excess about 8 times bigger than labeled strand.
Here we try to use dual color correlation method, fluorescence cross correlation spectroscopy, for detection the interaction between same length of double strand and single strand.
Materials and Method
Cy5- and Rhodamine Green (RG) labeled DNAs of 40 base length, and 20 base were used in this experiment as a model system.
Measurements were carried out with the concentration of 20 base, 40 base and control were 2x10-8M, 10-9M and 10-8M, respectively. FCS measurements were carried out wiht ConfoCor 2 (zeiss) with measurement timeof 40 sec.
Results
The translational diffusion time of both strands (plus and minus) is not sodifferent each other because the length of each strand is equal so that detection of the hybridization product (double strand) by using autocorrelation method (FCS) is very difficult. However, by using the calculation of cross correlation signals from FCCS between both Cy5- and RG, the process of hybridization was detected in a homogeneous solution without any physical separation method. Yield of double strand DNA was calculated about 50% for 40bp and 45% for 20bp with equation. On the other hand, in the case of hybridization between 40 base and 20 base, cross correlation signal was not detected because no complimentary sequence is contained. We now try to detect the formation of triplex DNA in solution by the new FCCS method.
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