STUDY FOR MECHANISM OF TRIPLEX USING SINGLE MOLECULES DETECTION

目的：在单分子检测时，用小体积荧光相关光谱分析水溶液中三螺旋DNA的形成过程。以荧光标记的寡核苷酸T15为探针DNA链，Homo Purin：Homo嘧啶DNA为靶链，制备三螺旋DNA。以改变探针DNA的平移扩散时间作为三链生成率的指标，在第一预算年确定FCS可作为三链的测定方法，并在第二预算年应用FCS方法分析溶液中三链的形成。方法：在荧光标记dUTP(Rho-dUTP)存在下，用聚合酶链式反应方法合成不同长度的荧光标记DNA(Rho-dUTP)。用FCS测定这些DNA的平移扩散时间，并以此为标准。为了与三链体的形成过程进行比较，我们测量了T15与互补链DNA的缔合过程。结论：双链DNA的平移扩散时间与棒状模型的理论值一致。由于三链DNA比双链DNA具有更多的刚性结构，我们可以用同样的方法用双组分模型分析三链DNA的平移扩散时间，结果表明三链DNA的形成速度慢于双螺旋结构。此外，在双链DNA的情况下，约80%的探针DNA与靶双链DNA结合，而添加的探针DNA链全部与靶DNA结合。靶DNA和/或探针DNA中错配序列的实验将在未来进行分析。

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.