Selective extraction of low-abundance BRAF V600E mutation from plasma, urine, and sputum using ion-tagged oligonucleotides and magnetic ionic liquids.

Sequence-specific DNA extractions have the potential to improve the detection of low-abundance mutations from complex matrices, making them ideal for circulating tumor DNA analysis during the early stages of cancer. Ion-tagged oligonucleotides (ITOs) are oligonucleotides modified with an allylimidazolium salt via thiolene click chemistry. The allylimidazolium-based tag allows the ITO-DNA duplex to be selectively captured by a hydrophobic magnetic ionic liquid (MIL). In this study, the selectivity of the ITO-MIL method was examined by extracting low abundance of the BRAF V600E mutation-a common single-nucleotide polymorphism associated with several different cancers-from diluted human plasma, artificial urine, and diluted artificial sputum. Quantitative polymerase chain reaction (qPCR) was not able to distinguish a 9% BRAF V600E standard (50 fg·μL BRAF V600E, 500 fg·μL wild-type BRAF) from the 100% wild-type BRAF (50 fg·μL) standard. However, introducing the ITO-MIL extraction prior to qPCR allowed for samples consisting of 0.1% BRAF V600E (50 fg·μL V600E BRAF, 50,000 fg·μL wild-type BRAF) to be distinguished from the 100% wild-type BRAF standard. Ion-tagged oligonucleotides (ITOs) are combined with magnetic ionic liquids (MILs) to extract low-abundance BRAF V600E mutation from diluted human plasma, artificial urine, and diluted artificial sputum. The ITO-MIL extraction prior to qPCR allowed for samples consisting of 0.1% BRAF V600E to be distinguished from the 100% wild-type BRAF standard.