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Biochem Biophys Res Commun. 2010 Apr 23;395(1):158-62. doi: 10.1016/j.bbrc.2010.03.167. Epub 2010 Mar 31.

The frequency of KRAS mutation detection in human colon carcinoma is influenced by the sensitivity of assay methodology: a comparison between direct sequencing and real-time PCR.

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  • 1Department of Surgery, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan.



Kirsten rat sarcoma (KRAS) gene mutations occur early in the progression of colorectal adenoma to carcinoma. The mutation status of the KRAS gene determines the benefits of molecular targeting drugs in patients with advanced colorectal cancer, although many methods are available to detect such mutations. The purpose of this study was to evaluate the influence of assay sensitivity on the detection frequency of mutated genes.


Colorectal tumors in 224 colorectal cancer patients were characterized for KRAS mutations using PCR amplification following by direct sequencing as well as a peptide nucleic acid (PNA)-clamp real-time PCR-based assay.


KRAS mutations were observed in 32.1% (72/224) patients by direct sequencing, and 43.3% (97/224) by PNA-clamp PCR. The chi-square test revealed that the difference in the frequency of KRAS mutations determined by direct sequencing and PNA-clamped PCR (threshold for 1% detection) was statistically significant (p<0.015).


Our data suggest that assay method sensitivity clearly influences the detection frequency of mutated genes. As more sensitive assays detect more mutated genes in clinical samples, this must be taken into consideration when determining KRAS gene status in clinical practice.

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