Use of fluorescent substrate 4-mup in the detection of biotin-labeled DNA probes

Abstract

To improve the sensitivity of detecting biotin-labeled DNA Probes, a new fluorescent substrate of alkaline phosphatase, 4-methylum belliferylphosphate (4-mup) was studied instead of conventional BCIP-NBT. The result of dot-blot hybridization demonstrates that this new substrate can be used for the colorimetric detection of biotin-labeled probes after hybridization to immobilized nucleic acids. The sensitivity is about one order of magnitude higher than that of BCIP-NBT system, and the time required for color development is very short, only about five min. It is suggested that the Bio-SA-Bio-AP-4-MUP colorimetric detection system can be widely used in gene diagnosis.

Keywords

biotin-labeled probe / nucleic acid hybridization / 4-methyl belliferylphosphate

References

[1] MillerJ A. Diagnostic DNA. Science News, 1984, 126: 104-104

[2] BerryA J, et al.. DNA probe for infectious diseases diagnosis. Medicine, 1984, 7(3): 62-62

[3] LearyI J, et al.. Rapid and sensitive colorimetric method for visualizing biotinlabeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bio-blots. Proc. Natl. Acad. Sci. USA, 1983, 50: 4045-4045

[4] FosterAC, et al.. Non-radioactive hybridization probes prepared by the chemical labeling of DNA and RNA with a novel reagent-photobiotin. Nucleic Acid Res, 1985, 13: 745-745

[5] Maniatis Tet al. Molecular cloning, 1982: 466–476