Allelic discrimination between circulating tumor DNA fragments enabled by a multiplex-qPCR assay containing...

Multiplex amplification of DNA can be highly valuable in circulating tumor DNA (ctDNA) analysis due to the sheer number of potential mutations. However, commercial ctDNA extraction methods struggle to preconcentrate low concentrations of DNA and require multiple sample handling steps. Recently, magnetic ionic liquids (MILs) have been used to extract DNA and were integrated with a quantitative polymerase chain reaction (qPCR). However, in previous studies, DNA could not be preconcentrated from plasma and only one fragment could be amplified per reaction. In this study, MILs were utilized as DNA extraction solvents and directly integrated into a multiplex-qPCR buffer to simultaneously amplify wild-type KRAS, G12S KRAS, and wild-type BRAF, three clinically-relevant genes whose mutation status can affect the success of anti-EGFR therapy. DNA was desorbed from the MIL solvent during a multiplex-PCR without having a significant effect on the amplification efficiency, and allelic discrimination of single-nucleotide polymorphisms could still be achieved. Enrichment factors over 35 for all three sequences were achieved from Tris buffer using the [N_8,8,8,Bz⁺][Ni(hfacac)₃^-]) and [P_6,6,6,14⁺][Ni(Phtfacac)₃^-] MILs. DNA could still be preconcentrated from 2-fold diluted human plasma using the [N_8,8,8,Bz⁺][Ni(hfacac)₃^-] MIL. Extractions from undiluted plasma were reproducible with the [P_6,6,6,14⁺][Ni(Phtfacac)₃^-] MIL although DNA was not preconcentrated with enrichment factors around 0.6 for all three fragments. Compared to commercial DNA extraction methods (i.e., silica-based spin columns and magnetic beads), the MIL-based extraction achieved higher enrichment factors in Tris buffer and plasma. The ability of the MIL-based dispersive liquid-liquid microextraction (DLLME) direct-multiplex-qPCR method to simultaneously achieve high enrichment factors of multiple DNA fragments from human plasma is highly promising in the field of ctDNA detection.