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- Title
High-sensitivity PCR method for detecting BRAF V600Emutations in metastatic colorectal cancer using LNA/DNA chimeras to block wild-type alleles.
- Authors
Chen, Dong; Huang, Jun-Fu; Xia, Han; Duan, Guang-Jie; Chuai, Zheng-Ran; Yang, Zhao; Fu, Wei-Ling; Huang, Qing
- 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 Δ C 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.
- Subjects
POLYMERASE chain reaction; GENETIC mutation; METASTASIS; CHIMERISM; ALLELES; EPIDERMAL growth factor receptors; COLON cancer treatment
- Publication
Analytical & Bioanalytical Chemistry, 2014, Vol 406, Issue 9/10, p2477
- ISSN
1618-2642
- Publication type
Academic Journal
- DOI
10.1007/s00216-014-7618-x