Multiple-labeling of oligonucleotide probes for in situ hybridization

Junzo Sasaki, Hitoshi Yamamoto, Takako Nomura, Junko Matsuura, Masaharu Seno, Eisuke F. Sato, Masayasu Inoue

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5 Citations (Scopus)

Abstract

We describe here a method to synthesize probes for in situ hybridization. This method provides more efficient incorporation of the reporter molecules such as 35S-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 I/Hind III-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.

Original languageEnglish
Pages (from-to)275-279
Number of pages5
JournalActa Histochemica et Cytochemica
Volume31
Issue number4
Publication statusPublished - 1998

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Keywords

  • Amelogenin
  • In situ hybridization
  • Oligonucleotide probe

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Endocrinology
  • Anatomy
  • Neuroscience(all)

Cite this

Sasaki, J., Yamamoto, H., Nomura, T., Matsuura, J., Seno, M., Sato, E. F., & Inoue, M. (1998). Multiple-labeling of oligonucleotide probes for in situ hybridization. Acta Histochemica et Cytochemica, 31(4), 275-279.