Fluorescence Detection of DNA Using a Novel Peroxidase Substrate, 4-(4-Hydroxyphenylcarbamoyl)butanoic Acid

Abstract

A phage DNA on nitrocellulose filters was quantified by a fluorometric assay using a biotin-labeled DNA probe and apacetamidophenol analog that yield a fluorescent product by enzymatic oxidation. Biotinylated A phage DNA (Hind III digested), used as a DNA probe, was prepared by diazobiotin, which reacts at the phosphate moiety of DNA. Peroxidase-labeled avidin was bound to biotin-labeled DNA, which was hybridized with ~. DNA on a nitrocellulose filter. The DNA was detected using a fluorogenic substrate for peroxidase, 4-(4-hydroxyphenylcarbamoyl)butanoic acid (HPBA) in the presence of hydrogen peroxide. The assay permits the determination of). phage DNA as low as 0.1 pg by a flow-through system. Keywords DNA, fluorometric assay, p-acetamidophenol analog, diazobiotin, fluorogenic substrateVarious kinds of techniques for detecting nucleotide have been developed so far, including Southern hybridization and dot blotting, which are the most common methods for detecting specific nucleotide sequences or measuring the amount of nucleotides. In order to analyze specific DNA, radio-labeled DNA is frequently used as a DNA probe. A technique using radio-labeled DNA probes, which employs mostly 32p labeled DNA, has been established with a high detection sensitivity. However, the method has a disadvantage in that the handling of radioactive materials is strictly limited because they are dangerous.In recent years, many alternative methods without using radioisotopes have been developed, such as colorimetric"2, fluorometric3'4 and chemiluminometric5,6 assays. Of these, nitro blue tetrazolium and 5-bromo-4-chloro-3-indoxylphosphate-4-toluidine salt as chromogens in enzymatic reactions are used for a highly sensitive colorimetric detection of specific DNA. Widely used reagents for chemiluminometric assays are luminol' and 4-methoxy-4-(3-phosphate phenyl)spiro[l,2-dioxethane-3,2-adamantane], disodium salt (AMPPD)8, which are the substrates for peroxidase and alkaline phosphatase, respectively. Chemiluminescence generated by enzymatic reactions can be detected with X-ray films, which also detect radiation generated from radioisotopelabeled DNA. Fluorometric labeling reagents are mostly applied to the determination of DNA sequencing by DNA sequencers.9"0 In enzymatic assays, biotinlabeled DNA probes are used in combination with an enzyme-labeled avidin, because of the strong binding ability of biotin for avidin. The biotin/avidin system has found wide applications in detecting biological materials. There are many reagents available for biotinylation of DNA, which include biotin hydrazide', photobiotin' 1 and diazobiotin.12In our previous reports12,13 we demonstrated that diazobiotin is a phosphate-reactive reagent for DNA labeling; also, the p-acetamidophenol analogs proved to be good fluorogenic substrates for peroxidase. Though 3-(4-hydroxyphenyl)propionic acid (HPPA) was a fairly sensitive reagent for measuring peroxidase activity14, pacetamidophenol analogs were twenty-times more sensitive than HPPA in the low peroxidase activ...