Biochemical and Biophysical Research Communications
Sensitive detection of FGFR3 mutations in bladder cancer and urine sediments by peptide nucleic acid-mediated real-time PCR clamping
Section snippets
Materials and methods
Samples and DNA extraction. Tumor tissues and preoperative voiding urine samples were obtained from 19 patients with bladder UCC who underwent TUR. Tumors were histologically staged according to the 1997 UICC TNM classification system [7] and graded according to the 1973 WHO classification system [8]. Voiding urine samples from 20 patients with chronic cystitis were assayed as negative controls. Genomic DNA was extracted by proteinase K digestion followed by phenol/chloroform extraction and
Validation of assay sensitivity by PNA-mediated real-time PCR clamping using serially diluted assay standards
We evaluated assay sensitivity using DNAs extracted from cell lines harboring FGFR3 mutations. In the analysis of exon 7, the proportion of UM-UC-14-derived DNA to normal genomic DNA, e.g., tumor cellularity, was adjusted to 100%, 10%, 1%, and 0%. Fifty nanograms of genomic DNA were used as templates for PNA-mediated real-time PCR clamping. The yield of the PCR product was indicated as the intensity of fluorescence emission from SYBR Green I dye bound to double-strand DNA fragments which
Discussion
The present study is the first report of the PNA-mediated real-time PCR clamping technique for detecting FGFR3 mutation. The assay can be performed by a simple one-step reaction in a single tube using SYBR® green I fluorescent dye. PNA-mediated real-time PCR clamping screens for the presence or absence of mutations; however, PCR products can be easily subjected to direct sequencing to identify the type of mutation.
The amounts of genomic DNA extractable from urine sediments are often trace,
Acknowledgments
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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