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High-sensitivity PCR method for detecting BRAF V600Emutations in metastatic colorectal cancer using LNA/DNA chimeras to block wild-type alleles

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Abstract

The response to epidermal growth factor receptor (EGFR)-targeted therapy in metastatic colorectal cancer (mCRC) is variable because of intra-tumor heterogeneity at the genetic level, and consequently, it is important to develop sensitive and selective assays to predict patient responses to therapy. Low-abundance BRAF V600E mutations are associated with poor response to treatment with EGFR inhibitors. We developed a method for the detection of BRAF V600E mutations in mCRC using real-time wild-type blocking PCR (WTB-PCR), in which a chimera composed of locked nucleic acids and DNA is incorporated to amplify the mutant allele at high efficiency while simultaneously inhibiting the amplification of wild-type alleles. Mixing experiments showed that this method is exquisitely sensitive, with detection of the mutated allele at a mutant/wild-type ratio of 1:10,000. To demonstrate the applicability of this approach for mCRC patients, we assessed the V600E mutations in 50 clinical cases of mCRC by real-time WTB-PCR. The percentage of patients with V600E mutation as determined by WTB-PCR (16 %, 8/50) was higher than by traditional PCR (10 %, 5/50), suggesting an increased sensitivity for WTB-PCR. By calculating the ΔC q for real-time traditional PCR, which amplifies all BRAF alleles, versus WTB-PCR, which selectively amplifies mutant BRAF, we demonstrated that among the V600E-positive mCRC patient samples, the percentage of BRAF DNA with the V600E mutation ranged from 0.05 to 52.32 %. In conclusion, WTB-PCR provides a rapid, simple, and low-cost method to detect trace amounts of mutated BRAF V600E gene.

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Acknowledgements

This work was supported in part by grants from the National High Technology Research and Development Program of China (National 863 Program, No. 2011AA02A121 and 2013AA020204), the National Key Technology R&D Program of China (No. 2012ZX10004801-003-006), the Major Project of Military Science and Technology “12th Five-Year Plan” (No. AWS11C001), the Preferential Foundation of Science and Technology Activities for Students Studying Abroad of Chongqing (No. YuLiuZhu201201), the Scientific Research Foundation for Returned Overseas Chinese Scholars.

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  1. Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing, 400038, China

    Dong Chen, Jun-Fu Huang, Han Xia, Zheng-Ran Chuai, Zhao Yang, Wei-Ling Fu & Qing Huang

  2. Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University, Chongqing, 400038, China

    Guang-Jie Duan

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  1. Dong Chen
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  2. Jun-Fu Huang
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Correspondence to Wei-Ling Fu or Qing Huang.

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Chen, D., Huang, JF., Xia, H. et al. High-sensitivity PCR method for detecting BRAF V600Emutations in metastatic colorectal cancer using LNA/DNA chimeras to block wild-type alleles. Anal Bioanal Chem 406, 2477–2487 (2014). https://doi.org/10.1007/s00216-014-7618-x

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