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

化学发光成像技术在许多研究领域得到了广泛的应用，人们迫切需要一种更加简便、灵敏的DNA检测方法。我们研究了利用非酶促荧光大分子探针检测DNA的新方法，其中许多鲁米诺和生物素分子被缀合到一个葡聚糖分子中，或三甲氧基苯基乙二醛（TMPG）标记的荧光鸟嘌呤和生物素被缀合到大的DNA中。利用生物素与亲和素之间的相互作用，这些探针可以与亲和素连接形成探针-链组装体。探针的化学发光强度随着并入葡聚糖中的鲁米诺的数目的增加而增加。含有2627个鲁米诺分子和385个生物素分子的右旋糖酐T2, 000（平均分子量，2，000 kDa）用于检测 关于我们 端粒DNA。该探针提供了在亚皮摩尔水平下的靶DNA的灵敏检测，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。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。TMPG的化学发光强度随DNA分子中鸟嘌呤碱基含量的增加而增加。因此，我们尝试了将端粒DNA与其cDNA结合在尼龙膜上的固定化杂交法，用CCD摄像机进行TMPG检测，使端粒DNA与生物素化的大DNA结合后，通过亲和素与生物素反应进行信号放大，获得了低至飞摩尔水平的高灵敏度。我们需要重新确认大DNA的生物标记。少

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