A FLUORESCENT DETECTION METHOD FOR DNA HYBRIDIZATION USING 2-HYDROXY-3-NAPHTHOIC ACID-2'-PHENYLANILIDE PHOSPHATE AS A SUBSTRATE FOR ALKALINE-PHOSPHATASE

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 (302 nm), bands containing as small as 70 fg (2.5 x 10(-20) mol) of DNA were detected after 2 hr of incubation. A higher sensitivity level of 10 fg was achieved in spot tests. This enables us to detect single copy genes with less than 1 mug of genomic DNA in southern blot hybridization. The reaction product is soluble in dimethylformamide, so that reprobing is possible after 1 min of immersion of membranes in dimethylformamide at room temperature. The HNPP method thus appears to be useful for detection of sub-picogram quantities of DNA.