Multiple-labeling of oligonucleotide probes for in situ hybridization

We describe here a method to synthesize probes for in situ hybridization. This method provides more efficient incorporation of the reporter molecules such as S-35-UTP or digoxigenin-UTP into the oligonucleotide probes than other methods. Two 99-base oligonucleotides complementary to each other were obtained as purified and lyophilized products (>99%). These oligonucleotides were designed as follows. The sequence of 77 bases derived from reported cDNA sequence in the literature was flanked by the restriction sites of EcoR I and Hind III (6 bases for each) with extended random sequences of 5 bases at both ends (total 99 bases). Both oligonucleotides were then annealed and digested with EcoR I and Hind III. The gel-purified EcoR II Hind Ill-cut DNA fragment was cloned into the pGEM4Z vector. The resultant plasmid DNA was linearized with EcoR I or Hind III and used as a template for the synthesis of labeled sense or antisense riboprobes. The amelogenin probes prepared by this method clearly distinguished the localized expression of mRNA when applied to in situ hybridization.