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ANALYSIS OF INTERACTION BETWEEN DNA STRANDS IN TRIPLE HELIX USING FLUORESCENCE CROSS CORRELATION SPECTROSCOPY.

使用荧光互相关光谱分析三重螺旋中 DNA 链之间的相互作用。

基本信息

批准号：
11680651
负责人：
KINJO Masataka
金额：
$ 2.05万
依托单位：
HOKKAIDO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

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 MethodCy5- 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.ResultsThe 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.

本研究的目的是用荧光相关光谱（FCCS）方法阐明DNA三螺旋中DNA链间的特征性相互作用，利用荧光相关光谱（FCS）中的自相关分析来监测杂交，为核酸研究开辟了新的灵敏度，然而，靶链的质量比必须超过标记链的8倍左右，本文尝试用双色相关法、荧光互相关法、材料与方法本实验以Cy 5-和罗丹明绿色（RG）标记的40碱基和20碱基的DNA为模型体系，在20碱基的浓度下进行测量，40碱和对照分别为2 × 10 ~（-8）M、10 ~（-9）M和10 ~（-8）M。结果两条链的平移扩散时间相差不大，因为每条链的长度相等，所以用自相关法（FCS）检测杂交产物（双链）是很困难的。然而，通过使用来自Cy 5-和RG两者之间的FCCS的互相关信号的计算，在均质溶液中检测杂交过程而无需任何物理分离方法。根据公式计算，40 bp和20 bp的双链DNA产率分别为50%和45%。另一方面，在40个碱基和20个碱基之间杂交的情况下，由于不含互补序列，所以没有检测到互相关信号。我们现在尝试用新的FCCS方法检测溶液中三链DNA的形成。