Detection of BRAF mutations in isolated melanoma cells in culture and in CTCs from melanoma patients.

<p>(A) PCR results indicating the presence or absence of the BRAF^V600E mutation. Cells exposed to the probe and rendered fluorescent (left inset) were isolated via capillary-based methods. Whole genome amplification (WGA) was performed on the DNA extracted from each cell, followed by quantitative polymerase chain reaction (qPCR) analysis using primers specific for the BRAF^V600E mutation. The presence of the mutation results in signal (Delta Rn, Y-axis) detectable by the 28th cycle and a curve of the characteristic “s” shape (left, arrow points to 28th cycle). The absence of the mutation results in no characteristic curve by the 28th cycle (right, arrow points to 28th cycle). (B) Isolation and genetic analysis for mutated BRAF^V600E in melanoma cells in culture and spiked in control blood. A375P (homozygous BRAF^V600E mutated), Mel624 (heterozygous BRAF^V600E mutated), and MeWo (homozygous BRAF WT) cells were isolated in culture and after being spiked into control blood using the capillary-based technique described. The DNA was extracted from each cell and subject to WGA, followed by qPCR analysis with primers specific for the BRAF^V600E mutation. In each case, the qPCR analysis accurately confirms the presence or absence of the BRAF^V600E mutation for each cell line. (C) Isolation of CTCs from patients and subsequent genetic analysis for BRAF mutation status. CTCs were isolated from an additional cohort of patients via capillary-based methods followed by DNA extraction, WGA, and qPCR analysis for BRAF^V600E. In each case, the BRAF mutation status of the isolated CTCs corresponded to that of the primary tumor.