A quantitative luminescence assay for nonradioactive nucleic acid probes.
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-methoxyspirol¿1,2-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.
Didenko
V V
VV
Huffington Center on Aging, Baylor College of Medicine, Houston, Texas, USA.
Hornsby
P J
PJ
D000218:Adamantane / Q000031:analogs & derivatives* / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002417:Cattle; D015342:DNA Probes / Q000032:analysis*; D004076:Digoxigenin / Q000032:analysis*; D008163:Luminescent Measurements; D018075:RNA, Complementary / Q000032:analysis; D013254:Steroid 17-alpha-Hydroxylase / Q000235:genetics; D013997:Time Factors
D015342:DNA Probes; D018075:RNA, Complementary; C071872:3-(2'-(spiro-5-chloroadamantane))-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane; D013254:Steroid 17-alpha-Hydroxylase; D000469:Alkaline Phosphatase; D004076:Digoxigenin; D000218:Adamantane