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

• Published in: Biochemical and biophysical research communications 2007-11, Vol.362 (4), p.865-871
• Main Authors: Miyake, Makito, Sugano, Kokichi, Kawashima, Kiyotaka, Ichikawa, Hiroki, Hirabayashi, Kaoru, Kodama, Tetsuro, Fujimoto, Hiroyuki, Kakizoe, Tadao, Kanai, Yae, Fujimoto, Kiyohide, Hirao, Yoshihiko
• Format: Article
• Language: English
• Subjects: DNA Mutational Analysis - methods; DNA Probes - genetics; FGFR3; Humans; Mutation; Peptide nucleic acid; Peptide Nucleic Acids - genetics; Polymorphism, Single Nucleotide - genetics; Real-time PCR; Receptor, Fibroblast Growth Factor, Type 3 - genetics; Receptor, Fibroblast Growth Factor, Type 3 - urine; Reverse Transcriptase Polymerase Chain Reaction - methods; Sensitivity and Specificity; Superficial bladder cancer; Urinalysis - methods; Urinary Bladder Neoplasms - genetics
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2007.08.092

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.