Abstract
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) ([P6,6,6,14+][Ni(hfacac)3−]), [P6,6,6,14+] tris(hexafluoroacetylaceto)colbaltate(II) ([Co(hfacac)3−]), [P6,6,6,14+] tris(hexafluoroacetylaceto)manganate(II) ([Mn(hfacac)3−]), or [P6,6,6,14+] 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 [P6,6,6,14+][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 [P6,6,6,14+][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.
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References
Jobling MA, Gill P. Encoded evidence: DNA in forensic analysis. Nat Rev Genet. 2004;5:739–51.
Schwarzenbach H, Hoon DSB, Pantel K. Cell-free nucleic acids as biomarkers in cancer patients. Nat Rev Cancer. 2011;11:426–37.
Bremen SU, Miller DN, Bryant JE, Madsen EL, Ghiorse WC. Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples. Appl Environ Microbiol. 1999;65(11):4715–24.
Schrader C, Schielke A, Ellerbroek L, Johne R. PCR inhibitors - occurrence, properties and removal. J Appl Microbiol. 2012;113(5):1014–26.
Patel R, Kvach JT, Mounts P. Isolation and restriction endonuclease analysis of mycobacterial DNA. J Gen Microbiol. 1986;132(2):541–51.
Wen J, Legendre LA, Bienvenue JM, Landers JP. Purification of nucleic acids in microfluidic devices. Anal Chem. 2008;80(17):6472–9.
Fan ZH, Mangru S, Granzow R, Heaney P, Ho W, Dong Q, et al. Dynamic DNA hybridization on a chip using paramagnetic beads. Anal Chem. 1999;71(21):4851–9.
Butler JM. U.S. initiatives to strengthen forensic science & international standards in forensic DNA. Forensic Sci Int Genet. 2015;18:4–20.
Hayashi S, Hamaguchi H. Discovery of a magnetic ionic liquid [bmim]FeCl4. Chem Lett. 2004;33(12):1590–1.
Del Sesto RE, McCleskey TM, Burrell AK, Baker GA, Thompson JD, Scott BL, et al. Structure and magnetic behavior of transition metal based ionic liquids. Chem Commun. 2008:447–9.
Krieger BM, Lee HY, Emge TJ, Wishart JF, Castner EW Jr. Ionic liquids and solids with paramagnetic anions. Phys Chem Chem Phys. 2010;12(31):8919–25.
Clark KD, Nacham O, Yu H, Li T, Yamsek MM, Ronning DR, et al. Extraction of DNA by magnetic ionic liquids: tunable solvents for rapid and selective DNA analysis. Anal Chem. 2015;87(3):1552–9.
Clark KD, Varona M, Anderson JL. Ion-tagged oligonucleotides coupled with a magnetic liquid support for the sequence-specific capture of DNA. Angew Chem Int Ed Engl. 2017;56(26):7630–3.
Clark KD, Purslow JA, Pierson SA, Nacham O, Anderson JL. Rapid preconcentration of viable bacteria using magnetic ionic liquids for PCR amplification and culture-based diagnostics. Anal Bioanal Chem. 2017;409(21):4983–91.
Merib J, Spudeit DA, Corazza G, Carasek E, Anderson JL. Magnetic ionic liquids as versatile extraction phases for the rapid determination of estrogens in human urine by dispersive liquid-liquid microextraction coupled with high-performance liquid chromatography-diode array detection. Anal Bioanal Chem. 2018. https://doi.org/10.1007/s00216-017-0823-7.
Yu H, Merib J, Anderson JL. Faster dispersive liquid-liquid microextraction methods using magnetic ionic liquids as solvents. J Chromatogr A. 2016;1463:11–9.
Chatzimitakos T, Binellas C, Maidatsi K, Stalikas C. Magnetic ionic liquid in stirring-assisted drop-breakup microextraction: proof-of-concept extraction of phenolic endocrine disrupters and acidic pharmaceuticals. Anal Chim Acta. 2016;910:53–9.
Clark KD, Yamsek MM, Nacham O, Anderson JL. Magnetic ionic liquids as PCR-compatible solvents for DNA extraction from biological samples. Chem Commun. 2015;51(94):16771–3.
Pandit KR, Nanayakkara IA, Cao W, Raghavan SR, White IM. Capture and direct amplification of DNA on chitosan microparticles in a single PCR-optimal solution. Anal Chem. 2015;87(21):11022–9.
Pierson SA, Nacham O, Clark KD, Nan H, Mudryk Y, Anderson JL. Synthesis and characterization of low viscosity hexafluoroacetylacetonate-based hydrophobic magnetic ionic liquids. New J Chem. 2017;41(13):5498–505.
Kreader CA. Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein. Appl Environ Microbiol. 1996;62(3):1102–6.
Taly V, Pekin D, Benhaim L, Kotsopoulos SK, Le Corre D, Li X, et al. Multiplex Picodroplet digital PCR to detect & KRAS mutations in circulating DNA from the plasma of colorectal cancer patients. Clin Chem. 2013;59(12):1722–31.
Behbahani M, Najafi F, Bagheri S, Bojdi MK, Salarian M, Bagheri A. Application of surfactant assisted dispersive liquid-liquid microextraction as an efficient sample treatment technique for preconcentration and trace detection of zonisamide and carbamazepine in urine and plasma samples. J Chromatogr A. 2013;1308(20):25–31.
Purohit HJ, Kapley A, Moharikar AA, Narde G. A novel approach for extraction of PCR-compatible DNA from activated sludge samples collected from different biological effluent treatment plants. J Microbiol Methods. 2003;52(3):315–23.
Wang H, Wang J, Zhang S. Binding Gibbs energy of ionic liquids to calf thymus DNA: a fluorescence spectroscopy study. Phys Chem Chem Phys. 2011;13(9):3906–10.
Stroun M, Anker P, Lyautey J, Lederrey C, Maurice PA. Isolation and characterization of DNA from the plasma of cancer patients. Eur J Cancer Clin Oncol. 1987;23(6):707–12.
Thierry AR, Mouliere F, El Messaoudi S, Mollevi C, Lopez-Crapez E, Rolet F, et al. Clinical validation of the detection of KRAS and BRAF mutations from circulating tumor DNA. Nat Med. 2014;20(4):430–5.
Devonshire AS, Whale AS, Gutteridge A, Jones G, Cowen S, Foy CA, et al. Towards standardisation of cell-free DNA measurement in plasma: controls for extraction efficiency, fragment size bias and quantification. Anal Bioanal Chem. 2014;406(26):6499–512.
Sparks AB, Struble CA, Wang ET, Song K, Oliphant A. Noninvasive prenatal detection and selective analysis of cell-free DNA obtained from maternal blood: evaluation for trisomy 21 and trisomy 18. Am J Obstet Gynecol. 2012;206(4):319.e1–9.
Stemmer C, Beau-Faller M, Pencreac’h E, Guerin E, Schneider A, Jaqmin D, et al. Use of magnetic beads for plasma cell-free DNA extraction: toward automation of plasma DNA analysis for molecular diagnostics. Clin Chem. 2003;49(11):1953–5.
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J.L.A. acknowledges funding from the Chemical Measurement and Imaging Program at the National Science Foundation (CHE-1709372).
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Emaus, M.N., Clark, K.D., Hinners, P. et al. Preconcentration of DNA using magnetic ionic liquids that are compatible with real-time PCR for rapid nucleic acid quantification. Anal Bioanal Chem 410, 4135–4144 (2018). https://doi.org/10.1007/s00216-018-1092-9
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DOI: https://doi.org/10.1007/s00216-018-1092-9