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Development of Super-Sensitive Technique for the Detection of Nucleic Acids with Luminescent Reagent

发光试剂检测核酸超灵敏技术的发展

基本信息

批准号：
14102031
负责人：
KAI Masaaki
金额：
$ 67.23万
依托单位：
Nagasaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (S)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-14102031/
关键词：
Dextran probe Sensitive detection Nucleic acid probe DNA analysis Development of technology Chemiluminescence Macromolecular probe Luminescent probe 発光試薬核酸高分子化合物プローブ解析

项目摘要

Chemiluminescence-imaging technology has been widely used in many fields of research, and a more facile and sensitive DNA-detection method has been desired for microanalysis. We studied novel detection methods of DNA utilizing non-enzymatic chemiluminescent macromolecular probes, in which many luminol and biotin molecules are conjugated into one dextran molecule, or trimethoxyphenylglyoxal (TMPG)-labeled chemiluminescent guanines and biotin are conjugated into large DNA. These probes can be tethered with avidin to form a probe-chained assembly based on the interaction between free avidins and biotins in the probe.Several chemiluminescent probes having luminol or isoluminol and biotin were synthesized using different sizes of dextran. The chemiluminescence intensity of the probes was increased with increased number of luminol incorporated into the dextran. DextranT2000 (average MW, 2,000 kDa) containing 2627 molecules of luminol and 385 molecules of biotin was used for the detection of … More telomere DNA. This probe afforded the sensitive detection of the target DNA at sub-picomole level, while problematic background signals were observed in a comparative study using a widely used horseradish peroxidase. The current method permitted a promising chemiluminescence-imaging detection of the target DNA.On the other hand, we utilized a unique chemical derivatization reagent, TMPG. This reagent reacted specifically with guanine bases in nucleic acids to produce quickly chemiluminescent derivatives under mild reaction conditions. TMPG gave an increasing CL intensity depending on the content of guanine base in the DNA molecule at picomole level. Thus we tried immobilized-hybridization assay of the telomere DNA binding to its cDNA on a nylon membrane.The TMPG detection with CCD camera allowed the high sensitivity as low as femtomole level of the telomere DNA after binding to biotinylated large DNA for signal amplification by the reaction between avidin and biotin. We are required to reconfirm the biotylation of large DNAs. Less

化学发光成像技术已广泛应用于许多研究领域，人们迫切需要一种更加简便、灵敏的dna检测方法来进行微量分析。我们研究了利用非酶化学发光大分子探针检测DNA的新方法，将许多发光鸟嘌呤和生物素分子偶联成一个葡聚糖分子，或将三甲氧基苯乙二醛（TMPG）标记的化学发光鸟嘌呤和生物素偶联成大的DNA。基于探针中游离亲和素和生物素之间的相互作用，这些探针可以与亲和素拴在一起形成探针链组装。用不同大小的葡聚糖合成了几种具有发光胺或异发光胺和生物素的化学发光探针。探针的化学发光强度随着加入葡聚糖的鲁米诺数量的增加而增加。DextranT2000（平均分子量，2,000 kDa）含有2627分子鲁米诺和385分子生物素，用于检测…该探针在亚皮摩尔水平上提供了对目标DNA的敏感检测，而在使用广泛使用的辣根过氧化物酶的比较研究中观察到有问题的背景信号。目前的方法允许对目标DNA进行有前途的化学发光成像检测。另一方面，我们使用了一种独特的化学衍生试剂TMPG。该试剂与核酸中的鸟嘌呤碱基特异性反应，在温和的反应条件下快速产生化学发光衍生物。在皮摩尔水平上，TMPG增加了DNA分子中鸟嘌呤碱基的含量。因此，我们尝试在尼龙膜上对端粒DNA与其cDNA结合进行固定化杂交试验。利用CCD相机进行TMPG检测，可以将端粒DNA与生物素化的大DNA结合后，通过亲和素与生物素的反应进行信号放大，灵敏度低至飞粒水平。我们需要重新确认大dna的生物基化。少