Quantitative detection of mutant alleles of the K-ras gene with minor groove binder-conjugated fluorogenic DNA probes

  • Authors:
    • Tetsuya Itabashi
    • Cihaya Maesawa
    • Michiro Uchiyama
    • Taro Higuchi
    • Tomoyuki Masuda
  • View Affiliations

  • Published online on: March 1, 2004     https://doi.org/10.3892/ijo.24.3.687
  • Pages: 687-696
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

Tumor-specific point mutations are stable biomarkers compared with tumor-specific mRNA expression, and are therefore useful to detect occult tumor cells. These mutations have never been used for real-time quantitative polymerase chain reaction (RQ-PCR) assays, because the ability of conventional probes to discriminate between wild-type and mutant alleles is poor. Recently, DNA probes with conjugated minor groove binder (MGB) have been developed. Because of their high melting temperature, these probes achieve high performance in detecting single nucleotide mismatches. Using the MGB technology, we developed a new RQ-PCR system for detecting occult tumor cells in patients with colorectal cancer (CRC), targeting K-ras point mutations. Sixteen MGB-conjugated DNA probes were designed for all previously reported K-ras mutations. The performance of these probes was examined with plasmid DNAs into which K-ras point mutations had been inserted, 32 cancer cell lines and 338 lymph nodes obtained from 15 CRC patients. Fifteen of the 16 MGB probes designed were useful for accurate quantitative assessment, and achieved high sensitivity (1/104-105 background cells) and high reproducibility (coefficients of variation <10%). Performance in discriminating single nucleotide mismatches was superior for MGB probes compared with non-MGB probes. We detected a micrometastasis (5.85/104 cells equivalent) in one (0.9%) of 110 lymph nodes obtained from 6 patients with K-ras mutations. There was no true false-positive result in 209 lymph nodes obtained from 9 patients without K-ras mutations. The MGB RQ-PCR assay targeting K-ras mutations is an accurate quantitative method for detecting occult tumor cells in CRC.

Related Articles

Journal Cover

March 2004
Volume 24 Issue 3

Print ISSN: 1019-6439
Online ISSN:1791-2423

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
APA
Itabashi, T., Maesawa, C., Uchiyama, M., Higuchi, T., & Masuda, T. (2004). Quantitative detection of mutant alleles of the K-ras gene with minor groove binder-conjugated fluorogenic DNA probes. International Journal of Oncology, 24, 687-696. https://doi.org/10.3892/ijo.24.3.687
MLA
Itabashi, T., Maesawa, C., Uchiyama, M., Higuchi, T., Masuda, T."Quantitative detection of mutant alleles of the K-ras gene with minor groove binder-conjugated fluorogenic DNA probes". International Journal of Oncology 24.3 (2004): 687-696.
Chicago
Itabashi, T., Maesawa, C., Uchiyama, M., Higuchi, T., Masuda, T."Quantitative detection of mutant alleles of the K-ras gene with minor groove binder-conjugated fluorogenic DNA probes". International Journal of Oncology 24, no. 3 (2004): 687-696. https://doi.org/10.3892/ijo.24.3.687