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A quantitative luminescence assay for nonradioactive nucleic acid probes

VV Didenko and PJ Hornsby

Huffington Center on Aging, Baylor College of Medicine, Houston, Texas, USA.

In histochemical work with digoxigenin- or biotin-labeled nucleic acid probes, reproducibility of in situ hybridization depends on accurate measurement of the amount of non-radioactive label being used. We describe a rapid and sensitive assay for nonradioactive label incorporated into nucleic acids employing a luminogenic substrate for alkaline phosphatase, CSPD (disodium 3-(4-methoxyspirol1,2-dioxetane- 3,2'-(5'-chloro)tricyclo [3.3.1.1(3,7)]decan-4-yl)phenyl phosphate). An alkaline phosphatase-antibody conjugate was bound to digoxigenin- labeled nucleic acids spotted on nylon membranes. Light emission from the reaction of the bound alkaline phosphatase with CSPD was measured with a luminometer. This method allows an accurate determination of digoxigenin incorporated into nucleic acid probes in the range of 0.5- 500 fmol of nonradioactive label.

Volume 44, Issue 6, pp. 657-660, 06/01/1996
Copyright © 1996 by The Histochemical Society


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[Abstract] [Full Text] [PDF]



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