A novel fluorescent method for detection of subpicogram quantities of nucleic acids

在重组DNA技术中，需要检测非常少量的核酸。在标准方案中，这是通过使用放射性标记的核酸作为探针来完成的。然而，放射性标记探针固有的缺点，如放射性分解引起的化学不稳定性、人员安全和处置问题、短的半衰期和放射自显影暴露时间等，限制了放射性标记探针在传统实验室中的应用。因此，需要有灵敏的非放射性同位素方法来检测核酸。近年来，一些实验室正在努力创新一种新的非放射性同位素方法，并提出了各种方法。其中，使用BCIP-Nitro BT或化学发光底物AMPPD作为底物的勃林格的地高辛-碱性磷酸酶法似乎是最可靠和敏感的方法。但该方法的灵敏度仍低于RI法。为了提高非ri法的灵敏度，我们在本研究中尝试为碱性磷酸酶合成荧光底物，这种底物在水解后产生不溶性的高荧光产物。结果发现HNPP（3-羟基-n -2-联苯基-2-萘甲酰胺磷酸）是合成的86种荧光染料中最有用的底物。有了这种底物，通过斑点测试可以提取小到10fg的DNA，通过southern印迹可以提取70fg的DNA。该方法在分辨率、速度、反应监测、数据存储、重测和试剂稳定性等方面优于现有的任何技术。

In recombinant DNA techniques, it is required to detect a very small amount of nucleic acids. In standard protocols this is accomplished by the use of radio-labeled nucleic acids as probes. However, the inherent drawbacks of radiolabeled probes, such as chemical lability due to radiolytic decomposition, personnel safety and disposal problems, short half-life and duration of autoradiographic exposure limit the usefulness of radio-labeled probes in conventional laboratories. It is therefore desirable to have sensitive, non-radioisotope methods for detecting nucleic acids. Recontly, efforts have been made in a number of laboratories to innovate a new non-radioisotopic methods and a variety of methods have been proposed. Among these, the Boehringer's Digoxigenin-alkaline phosphosphatase method utilizing either BCIP-Nitro BT or a chemiluminescent substrate, AMPPD as a substrate appears to be most reliable and sensitive method. However, the method is still lower in sensitivity than the RI method. To improve the sensitivity of thenon-RI method, we have attempted in this profect to synthesize fluorogenic substrates for alkalien phosphatase which upon hydrolysis yield insoluble, highly fluorescent products. As a result, HNPP (3-hydroxy-N-2-biphenylyl-2-naphthalenecarboxamide phosphate) was found to be the most useful substrate among a total of 86 fluorochromes synthesized. With this substrate, it is possible to detact as small as 10fg of DNA by spot tests, and 70fg of DNA by southern blots. The method ustilizing HNPP is superior to any available techniques with respect to resolution, speed, ease of reaction monitoring, data storage, reprobing and stability of reagents.