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实现的。然而，放射性标记探针的固有缺陷，如辐射分解引起的化学不稳定性、人员安全和处置问题、半衰期短和放射自显影曝光时间短等限制了放射性标记探针在常规实验室中的应用。因此，人们希望有灵敏的、非放射性的检测核酸的方法。与此同时，许多实验室都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，以BCIP-Nitro BT或化学发光底物AMPPD为底物的Boehringer‘s地高辛-碱性磷酸酶法最为可靠和灵敏。然而，该方法的灵敏度仍然低于RI方法。为了提高NON-RI方法的灵敏度，我们尝试合成了烷基磷酸酶的荧光底物，这种底物在水解时会产生不溶的、强荧光的产物。结果发现，在合成的86种荧光基团中，(3-hydroxy-N-2-biphenylyl-2-naphthalenecarboxamide磷酸盐是最有用的底物。有了这种底物，通过斑点试验可以分离出小到10fg的DNA，通过Southern杂交可以分离出70fg的DNA。利用HNPP的方法在分辨率、速度、反应监测、数据存储、重复探测和试剂的稳定性方面优于任何现有的技术。

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.