Sequence-specific eDNA extraction using hydrophobic magnetic ionic liquids attached with oligonucleotide strand

Isolation of high-purity nucleic acids, especially sequence-specific DNA, from complex samples is critical to the downstream nucleic acid analysis. In this work, an oligonucleotide strand-attached magnetic ionic liquid (OSMIL) was designed and prepared for DNA extraction. The attached oligonucleotide strand has a sequence complementary to that of a specific DNA to be extracted. The OSMIL has good hydrophobicity and magnetic response properties. At the extraction temperature, OSMIL was in a liquid state, which was favorable for maximizing the adsorption of DNA; while at the separation temperature, OSMIL was in a solid state (with an average particle size of 897 nm) and could be attracted by an external magnet in 3s, which was favorable for the separation and recovery of DNA. The sequence-specific DNA extraction process with OSMIL is simple and fast. After extraction, the DNA-enriched OSMILs were quickly attracted and separated by an external magnetic field. The extracted DNA was evaluated by a NanoDrop (wavelength detection at 260-280 nm) and the OSMIL can be recycled and reused. The enrichment factor was 0.81. Through single-factor experimental analysis, the effects of OSMIL extraction volume, thermal excitation temperature, thermal excitation time, pH, and other factors on the DNA extraction process were systematically investigated. The RSD of repeatability experiment was 1.19% (n = 3), showing the method has good repeatability. The extraction method presented here has been shown to extract DNA with specific sequences from mixtures containing DNA of different sequences and from mixtures containing proteins, respectively. In addition, the OSMIL has been applied to extract target environmental DNA with specific sequences from different water environments with high extraction efficiency. In the long run, OSMIL has great potential for identifying existing organisms in environmental samples or exploring unknown organisms.