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

doi:10.1016/j.bbrc.2007.08.092

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

Research output: Contribution to journal › Article › peer-review

29 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 language	English
Pages (from-to)	865-871
Number of pages	7
Journal	Biochemical and Biophysical Research Communications
Volume	362
Issue number	4
DOIs	https://doi.org/10.1016/j.bbrc.2007.08.092
Publication status	Published - 2007 Nov 3
Externally published	Yes

Keywords

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

ASJC Scopus subject areas

Biophysics
Biochemistry
Molecular Biology
Cell Biology

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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

Sensitive detection of FGFR3 mutations in bladder cancer and urine sediments by peptide nucleic acid-mediated real-time PCR clamping. / Miyake, Makito; Sugano, Kokichi; Kawashima, Kiyotaka; Ichikawa, Hiroki; Hirabayashi, Kaoru; Kodama, Tetsuro; Fujimoto, Hiroyuki; Kakizoe, Tadao; Kanai, Yae; Fujimoto, Kiyohide; Hirao, Yoshihiko.

In: Biochemical and Biophysical Research Communications, Vol. 362, No. 4, 03.11.2007, p. 865-871.

Research output: Contribution to journal › Article › peer-review

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, vol. 362, no. 4, pp. 865-871. https://doi.org/10.1016/j.bbrc.2007.08.092

Miyake, Makito ; Sugano, Kokichi ; Kawashima, Kiyotaka ; Ichikawa, Hiroki ; Hirabayashi, Kaoru ; Kodama, Tetsuro ; Fujimoto, Hiroyuki ; Kakizoe, Tadao ; Kanai, Yae ; Fujimoto, Kiyohide ; Hirao, Yoshihiko. / Sensitive detection of FGFR3 mutations in bladder cancer and urine sediments by peptide nucleic acid-mediated real-time PCR clamping. In: Biochemical and Biophysical Research Communications. 2007 ; Vol. 362, No. 4. pp. 865-871.

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

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.",

keywords = "FGFR3, Mutation, Peptide nucleic acid, Real-time PCR, Superficial bladder cancer",

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

note = "Funding Information: This work was supported in part by Grants-in-Aid for Cancer Research and by the Award of a Research Resident Fellowship from the Foundation for Promotion of Cancer Research (Japan) for the 3rd Term Comprehensive 10-Year Strategy for Cancer Control.",

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AU - Ichikawa, Hiroki

AU - Hirabayashi, Kaoru

AU - Kodama, Tetsuro

AU - Fujimoto, Hiroyuki

AU - Kakizoe, Tadao

AU - Kanai, Yae

AU - Fujimoto, Kiyohide

AU - Hirao, Yoshihiko

N1 - Funding Information: This work was supported in part by Grants-in-Aid for Cancer Research and by the Award of a Research Resident Fellowship from the Foundation for Promotion of Cancer Research (Japan) for the 3rd Term Comprehensive 10-Year Strategy for Cancer Control.

PY - 2007/11/3

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N2 - 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.

AB - 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.

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

Abstract

Keywords

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