A fluorescent detection method for DNA hybridization using 2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate as a substrate for alkaline phosphatase.

Abstract

Among a total of 60 fluorochromes synthesized, 2-hydroxy-3-naphthoic acid-2′-phenylanilide phosphate (HNPP) was found to be the most suitable for detection of membrane-bound DNA; Hind III-fragments of lambda DNA were electrophoresed, transferred to nylon membrane, hybridized with digoxigenin-labeled probes, treated with phosphatase-labeled antibody and then incubated with HNPP as a substrate. The fluorescent reaction product is insoluble in water and precipitates in situ. Under UV illumination (302nm), bands containing as small as 70fg (2.5×10-20mol) of DNA were detected after 2hr of incubation. A higher sensitivity level of 10fg was achieved in spot tests. This enables us to detect single copy genes with less than 1μg of genomic DNA in southern blot hybridization. The reaction product is soluble in dimethylformamide, so that reprobing is possible after 1min of immersion of membranes in dimethylformamide at room temperature. The HNPP method thus appears to be useful for detection of subpicogram quantities of DNA.