摘要
研究目的是拓展基激复合物荧光探针方法在检测指定序列DNA中的应用。实验选择细胞色素P450 CYP2C9基因中容易发生突变的包含24个碱基的基因片段作为靶点DNA，选择两个分开的与靶点碱基相对应的包含12碱基的寡核苷酸作为探针，将荧光集团连接到探针末端3′或5′磷酸基上形成荧光探针，两个荧光探针与靶点DNA杂交后自动装备基激复合物荧光系统，实验考察芘的新衍生物与不同荧光团配对、荧光团连接不同位置、不同激发波长对基激复合物形成及发射光谱的影响，芘的新衍生物探针形成的基激复合物在505 nm的特征发射光谱最强，伴随着单体荧光的猝灭和大约120～130 nm的Stokes位移，大大减少了DNA检测过程中的背景干扰，灵敏度高，而且这对荧光物质形成的基激复合物对所处的空间位置及微环境非常敏感，可尝试用于基因遗传多态性的识别。
The present research was to develop the exciplex-based fluorescence detection of DNA. A SNP-containing region of eytoehrome P450 2C9 DNA systems was evaluated to define some of the structural and associated requirement of this new class of exeiplex-formed probe, and a 24-base target was selected which contains singlevnueleotide polymorphisms （SNP） in genes coding for cytoehrome P450. The two probes were all 12-base to give coverage of a 24-base target region to ensure specificity within the human genome. Exciplex partners used in this study were prepared using analogous phosphoramide attaehment to the 3＇- or 5＇-phosphate group of the appropriate oligonucleotide probes. The target effectively assembled its own detector by hybridization from components which were non-fluorescent at the detection wavelength, leading to the huge improvement in terms of deereased background. This research provides details of the effects of different partner, position of partners and different excitation wave- lengths for the split-oligonucleotide probe system for exciplex-based fluorescence detection of DNA. This study demonstrates that the emission intensity of the excimer formed by new pyrene derivative is the highest in these excimer and exeiplex, and the exeimer is easy to be formed and not sensitive to the position of partners. However the exeiplex formed by the new pyrene derivative and naphthalene emitted strongly at -505 nm with large Stokes shifts （120-130 nm）, and the monomer emission at 390 and 410 nm is nearly zero. Excitation wavelength of 400 nm is the best for Ie505/Im410 （exciplex emission at 505 nm/monomer emission at 410 nm） of the exciplex. This method features lower background and high sensitivity. Moreover the exeiplex is sensitive to the steric factor, different position of partners and microenvironment, so this exciplex system is promising and could be tried to identify the SNP genes.
出处
《光谱学与光谱分析》
SCIE
EI
CAS
CSCD
北大核心
2009年第7期1962-1966,共5页
Spectroscopy and Spectral Analysis
基金
国家自然科学基金项目(30600052)
2006新世纪优秀人才项目(NCET-06-0329)资助
