A Novel Fluorogenic Substrate for the Use of Nucleic Acid Hybridization

The phosphates derived from newly synthesized 3'-O-aralkyl-5-(4-biphenylcarboxamido)fluorescein derivatives were examined for the alkaline phosphatase-linked fluorescence assay of the membrane-bound DNA. lambda DNA was detectable to the amount of 5 fg in the assay using phosphorylated 5-(4-biphenylcarboxamido)-3'-O-(1-naphthyl)methylfluorescein (BNFP). The spots gave distinguishably clear fluorescence without diffusion and non-specific adsorption. In the Southern blot hybridization, 0.1 pg of DNA could be detected. Detection of two different DNAs on a single blot was successfully performed by using two different fluorogenic phosphates, Phosphrorilated N-(2-biphenyl)-3-hydroxy-2-naphthalenecarboxamide (HNPP) and BNFP. The fluorescein derivatives were also examined as substrates for horse radish peroxidase.