Preconcentration of DNA using magnetic ionic liquids that are compatible with real-time PCR for rapid nucleic acid quantification

摘要

Nucleic acid extraction and purification represents a major bottleneck in DNA analysis. Traditional methods for DNA purification often require reagents that may inhibit quantitative polymerase chain reaction (qPCR) if not sufficiently removed from the sample. Approaches that employ magnetic beads may exhibit lower extraction efficiencies due to sedimentation and aggregation. In this study, four hydrophobic magnetic ionic liquids (MILs) were investigated as DNA extraction solvents with the goal of improving DNA enrichment factors and compatibility with downstream bioanalytical techniques. By designing custom qPCR buffers, we directly incorporated DNA-enriched MILs including trihexyl(tetradecyl)phosphonium tris(hexafluoroacetylaceto)nickelate(II) (P-6,P-6,P-6,P-14 (+)Ni(hfacac)(3) (-)), P-6,P-6,P-6,P-14 (+) tris(hexafluoroacetylaceto)colbaltate(II) (Co(hfacac)(3) (-)), P-6,P-6,P-6,P-14 (+) tris(hexafluoroacetylaceto)manganate(II) (Mn(hfacac)(3) (-)), or P-6,P-6,P-6,P-14 (+) tetrakis(hexafluoroacetylaceto)dysprosate(III) (Dy(hfacac)(4) (-)) into reaction systems, thereby circumventing the need for time-consuming DNA recovery steps. Incorporating MILs into the reaction buffer did not significantly impact the amplification efficiency of the reaction (91.1). High enrichment factors were achieved using the P-6,P-6,P-6,P-14 (+)Ni(hfacac)(3) (-) MIL for the extraction of single-stranded and double-stranded DNA with extraction times as short as 2 min. When compared to a commercial magnetic bead-based platform, the P-6,P-6,P-6,P-14 (+)Ni(hfacac)(3) (-) MIL was capable of producing higher enrichment factors for single-stranded DNA and similar enrichment factors for double-stranded DNA. The MIL-based method was applied for the extraction and direct qPCR amplification of mutation prone-KRAS oncogene fragment in plasma samples.