Figure 3.

Peptide-based affinity isolation improves the detection of mutant DNA from ctDNA and EVs in low-copy number samples. (A) Recovery of decreasing amounts of mutation-positive extracellular vesicles (EVs) from healthy donor (HD) plasma samples with peptide affinity (PA) isolation and cell-free circulating nucleic acid isolation protocol (CF). Following DNA extraction, BRAF^V600E gene copies were detected by allele-specific quantitative PCR (AS-QPCR) and expressed as arbitrary units (AU) on a logarithmic scale. (B) BRAF^V600E allelic frequency was calculated after quantifying BRAF^WT gene copies by AS-QPCR and expressed as % on a logarithmic scale. Data are representative of three independent experiments. (C) Recovery of mutant DNA from both BRAF^V600E-positive EVs and KRAS^G12S-positive cfDNA by PA and CF isolation. 2.5 µg of BRAF^V600E-positive EVs and 10 nanograms of KRAS^G12S-positive cfDNA were spiked into HD plasma and isolated by PA and CF isolation. Following DNA extraction, mutant copies were quantified by digital PCR (dPCR). BRAF^V600E gene copies and KRAS^G12S gene copies were considered as total mutant copies in samples where cfDNA and EVs were co-spiked in the same plasma. Results are representative of three independent experiments.