title: High-sensitivity PCR method for detecting BRAF V600E mutations in metastatic colorectal cancer using LNA/DNA chimeras to block wild-type alleles.
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.
authors: Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing, 400038, China.
authors: Dong
authors: D
authors: Chen
authors: Jun-Fu
authors: JF
authors: Huang
authors: Han
authors: H
authors: Xia
authors: Guang-Jie
authors: GJ
authors: Duan
authors: Zheng-Ran
authors: ZR
authors: Chuai
authors: Zhao
authors: Z
authors: Yang
authors: Wei-Ling
authors: WL
authors: Fu
authors: Qing
authors: Q
authors: Huang
mesh_terms: D000483:Alleles; D001483:Base Sequence; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000201:enzymology* / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D009696:Nucleic Acids / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*
chemical_list: D009696:Nucleic Acids; D004247:DNA; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf