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Allelic discrimination between circulating tumor DNA fragments enabled by a multiplex-qPCR assay containing DNA-enriched magnetic ionic liquids

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  • Additional Information
    • Affiliation:
      Department of Chemistry, Iowa State University, Ames, IA, 50011, USA
    • Keywords:
      DNA extraction
      Cell-free DNA
      Multiplex PCR
      Magnetic ionic liquid
    • Abstract:
      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 [N8,8,8,Bz+][Ni(hfacac)3-]) and [P6,6,6,14+][Ni(Phtfacac)3-] MILs. DNA could still be preconcentrated from 2-fold diluted human plasma using the [N8,8,8,Bz+][Ni(hfacac)3-] MIL. Extractions from undiluted plasma were reproducible with the [P6,6,6,14+][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.
    • Abstract:
      @@@@Highlights •Custom multiplex-qPCR assays were developed to contain magnetic ionic liquid (MIL) solvents.•Circulating tumor DNA (ctDNA) was preconcentrated from plasma matrices with the MILs.•DNA-enriched MIL was added to the multiplex-qPCR assay to desorb captured DNA.•Allelic discrimination and good PCR efficiency was achieved with the MIL-multiplex-qPCR assay.•Commercial kits poorly preconcentrated DNA due to high desorption volumes.
    • ISSN:
      0003-2670
    • Accession Number:
      10.1016/j.aca.2020.04.078
    • Accession Number:
      S0003267020305110
    • Copyright:
      © 2020 Elsevier B.V. All rights reserved.
  • Citations
    • ABNT:
      EMAUS, M. N.; ANDERSON, J. L. Allelic discrimination between circulating tumor DNA fragments enabled by a multiplex-qPCR assay containing DNA-enriched magnetic ionic liquids. Analytica Chimica Acta, [s. l.], v. 1124, p. 184–193, 2020. DOI 10.1016/j.aca.2020.04.078. Disponível em: http://search.ebscohost.com/login.aspx?direct=true&site=eds-live&db=edselp&AN=S0003267020305110. Acesso em: 20 out. 2020.
    • AMA:
      Emaus MN, Anderson JL. Allelic discrimination between circulating tumor DNA fragments enabled by a multiplex-qPCR assay containing DNA-enriched magnetic ionic liquids. Analytica Chimica Acta. 2020;1124:184-193. doi:10.1016/j.aca.2020.04.078
    • APA:
      Emaus, M. N., & Anderson, J. L. (2020). Allelic discrimination between circulating tumor DNA fragments enabled by a multiplex-qPCR assay containing DNA-enriched magnetic ionic liquids. Analytica Chimica Acta, 1124, 184–193. https://doi.org/10.1016/j.aca.2020.04.078
    • Chicago/Turabian: Author-Date:
      Emaus, Miranda N., and Jared L. Anderson. 2020. “Allelic Discrimination between Circulating Tumor DNA Fragments Enabled by a Multiplex-QPCR Assay Containing DNA-Enriched Magnetic Ionic Liquids.” Analytica Chimica Acta 1124 (August): 184–93. doi:10.1016/j.aca.2020.04.078.
    • Harvard:
      Emaus, M. N. and Anderson, J. L. (2020) ‘Allelic discrimination between circulating tumor DNA fragments enabled by a multiplex-qPCR assay containing DNA-enriched magnetic ionic liquids’, Analytica Chimica Acta, 1124, pp. 184–193. doi: 10.1016/j.aca.2020.04.078.
    • Harvard: Australian:
      Emaus, MN & Anderson, JL 2020, ‘Allelic discrimination between circulating tumor DNA fragments enabled by a multiplex-qPCR assay containing DNA-enriched magnetic ionic liquids’, Analytica Chimica Acta, vol. 1124, pp. 184–193, viewed 20 October 2020, .
    • MLA:
      Emaus, Miranda N., and Jared L. Anderson. “Allelic Discrimination between Circulating Tumor DNA Fragments Enabled by a Multiplex-QPCR Assay Containing DNA-Enriched Magnetic Ionic Liquids.” Analytica Chimica Acta, vol. 1124, Aug. 2020, pp. 184–193. EBSCOhost, doi:10.1016/j.aca.2020.04.078.
    • Chicago/Turabian: Humanities:
      Emaus, Miranda N., and Jared L. Anderson. “Allelic Discrimination between Circulating Tumor DNA Fragments Enabled by a Multiplex-QPCR Assay Containing DNA-Enriched Magnetic Ionic Liquids.” Analytica Chimica Acta 1124 (August 8, 2020): 184–93. doi:10.1016/j.aca.2020.04.078.
    • Vancouver/ICMJE:
      Emaus MN, Anderson JL. Allelic discrimination between circulating tumor DNA fragments enabled by a multiplex-qPCR assay containing DNA-enriched magnetic ionic liquids. Analytica Chimica Acta [Internet]. 2020 Aug 8 [cited 2020 Oct 20];1124:184–93. Available from: http://search.ebscohost.com/login.aspx?direct=true&site=eds-live&db=edselp&AN=S0003267020305110