A Novel Fluorogenic Substrate for the Use of Nucleic Acid Hybridization

Abstract

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. λDNA 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 nonspecific 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.