Sensitive detection of FGFR3 mutations in bladder cancer and urine sediments by peptide nucleic acid-mediated real-time PCR clamping

Makito Miyake, Kokichi Sugano, Kiyotaka Kawashima, Hiroki Ichikawa, Kaoru Hirabayashi, Tetsuro Kodama, Hiroyuki Fujimoto, Tadao Kakizoe, Yae Kanai, Kiyohide Fujimoto, Yoshihiko Hirao

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28 Citations (Scopus)

Abstract

Somatic mutations of the fibroblast growth factor receptor 3 (FGFR3) gene were detected by peptide nucleic acid (PNA)-mediated real-time PCR clamping. Mutation was detected in negative control containing only wild-type DNA due to a misincorporation of dNTPs to PNA binding sites when the amount of template DNA was decreased to 1 ng. Thus, the amount of template DNA was critical determinant of the assay sensitivity in PNA-mediated PCR clamping. Assay conditions were optimized to detect FGFR3 mutations in exons 7, 10, and 15, at a concentration of more than 1% mutated DNA using 50 ng of genomic DNA as the template. Mutations were detected in 12 of 13 (92.3%) tumor tissues and 11 of 13 (84.6%) urine samples from patients with superficial bladder cancer, while no mutations were detected in tissues and/or urine samples from patients with muscle-invasive bladder cancer or chronic cystitis.

Original languageEnglish
Pages (from-to)865-871
Number of pages7
JournalBiochemical and Biophysical Research Communications
Volume362
Issue number4
DOIs
Publication statusPublished - 2007 Nov 3
Externally publishedYes

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Keywords

  • FGFR3
  • Mutation
  • Peptide nucleic acid
  • Real-time PCR
  • Superficial bladder cancer

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Miyake, M., Sugano, K., Kawashima, K., Ichikawa, H., Hirabayashi, K., Kodama, T., Fujimoto, H., Kakizoe, T., Kanai, Y., Fujimoto, K., & Hirao, Y. (2007). Sensitive detection of FGFR3 mutations in bladder cancer and urine sediments by peptide nucleic acid-mediated real-time PCR clamping. Biochemical and Biophysical Research Communications, 362(4), 865-871. https://doi.org/10.1016/j.bbrc.2007.08.092