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简便、更灵敏的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，将多个鲁米诺和生物素分子连接到一个葡聚糖分子中，或者将三甲氧基苯乙二醛(TMPG)标记的化学发光鸟嘌呤和生物素连接成大DNA。根据游离型亲和素与生物素之间的相互作用，这些探针可以与亲和素结合形成探针-链式组合体。用不同大小的葡聚糖合成了鲁米诺或异鲁米诺-生物素化学发光探针。随着葡聚糖中鲁米诺掺杂量的增加，探针的化学发光强度增强。使用含有2627个鲁米诺分子和385个生物素分子的右旋糖苷T2000(平均分子量2,000 kDa)来检测…更多端粒DNA。该探针在亚皮摩尔水平上提供了对目标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了有问题的背景信号。目前的方法允许一个有前景的化学发光成像检测目标DNA。另一方面，我们使用了一种独特的化学衍生化试剂，TMPG。该试剂与核酸中的鸟嘌呤碱基发生特异反应，在温和的反应条件下快速生成化学发光衍生物。在微克分子水平上，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，我们尝试了端粒DNA与其在尼龙膜上的结合的固定化杂交分析。TMPG用CCD摄像机检测，使端粒DNA与生物素化的大DNA结合后，通过亲和素和生物素的反应进行信号放大，其灵敏度低至毫微摩尔水平。我们需要重新确认大DNA的生物甲基化。较少

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