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

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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.