DEVELOPMENT OF FLUORESCENCE CORRELATION SPECTROSCOPY SYSTEM AIM AT MOLECULAR DIAGNOSIS FOR MASS EXAMINATION

聚合酶链式反应和改良的聚合酶链式反应方法，如逆转录酶，已经被用来检测特定的基因和基因组信息。然而，传统的聚合酶链式反应需要进行酶促反应、产物分离和检测等步骤，采用非对称聚合酶链式反应的方法进行单链DNA扩增。我们关注的不是单链DNA(SsDNA)，而是另一种产品，双链DNA(DsDNA)。在扩增过程中，当荧光标记的引物浓度低于另一对引物时，可以预期反应产物为荧光双链DNA和非荧光单链DNA。在理想的条件下，所有的荧光标记的引物(低浓度的引物)在适当的PCR循环次数后都被整合到dsDNA中。从荧光分子的角度出发，在基于荧光标记的不对称聚合酶链式反应(FA-PCR)中，将短的单链DNA延长为长的双链DNA，在均相条件下用荧光相关光谱仪(FCS)对扩增产物进行检测。FCS提供了分子在体积元素中的平均数目和分子的扩散常数。在本项目中，我们使用FCS作为一种简单的工具来检测特定基因组和序列，来测定FA-PCR中几种DNA的产率。结果表明，FCS和FA-PCR的结合可以在初始浓度为25微滴的溶液中检测到单拷贝水平的目的基因，而不需要使用凝胶电泳等任何分离或纯化方法。该系统由载样台和数据评价计算机系统组成。

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.