MOLECULAR HYBRIDIZATION PROBES MOLECULAR HYBRIDIZATION PROBES PREPARED WITH 4-AMINOOXYBUTYLAMINE

A versatile method is described for preparing nonradioactive DNA probe s for molecular hybridization. The method is based on the transaminati on reaction of double-stranded DNA with 4-aminooxybutylamine (ABA). To optimize the procedure for obtaining stable and sensitive hybridizati on probes, time of modification, pH, and reaction temperature were var ied. The optimal reaction conditions allowed the preparation of nonrad ioactive-labeled DNA probes that met demands for optimal length, modif ication degree, stability, and sensitivity. The use of 4-aminooxybutyl amine as a bifunctional reagent for DNA modification allowed the possi bility of choosing an appropriate reporter group: biotin or one of the fluorochromes. For probes carrying biotin, a high specificity and hig h sensitivity detection limit of 1 pg of target DNA were demonstrated. In addition, the applicability of probes carrying fluorochromes for m ulticolor direct fluorescence in situ hybridization was described.