Ion‐Tagged Oligonucleotides Coupled with a Magnetic Liquid Support for the Sequence‐Specific Capture of DNA

Abstract

The isolation of specific nucleic acid sequences is am ajor bottlenecki nm olecular diagnostics.M agnetic beads/ particles are typically used as solid supports for the capture of DNAtargets to improve sample throughput but aggregate over time resulting in lower capture efficiency and obstruction of liquid handling devices.H erein, we describe ap article-free approach to sequence-specific DNAe xtraction using am agnetic liquid support and ion-tagged oligonucleotide (ITO) probes.ITO conjugates were synthesized with the highest yields ever achieved for the radical thiol-ene coupling of as ubstrate and oligonucleotide.I na ddition to distinguishing nucleotide mismatches,the ITOand magnetic liquid-based approach was more sensitive than acommercial magnetic bead-based method for the capture of target DNAf rom ap ool of interfering genomic DNA.

Ionic liquids: prospects for nucleic acid handling and delivery

“…Another promising application of ILs is nucleic acid extraction and preconcentration; IL-assisted extraction of DNA from aqueous media provides suitable templates for PCR and real-time PCR ( 114–117 ). Sequence-specific extraction of DNA also can be achieved ( 106–108 , 118 , 119 ). A separate, though related branch of studies concerns the application of ILs in targeted analysis of DNA sequences ( 104 ), including detection of mismatched base pairs which can be of use in diagnostics and gene therapy ( 68 ), and electrochemical biosensors for nucleic acids that operate in very small volumes ( 120–129 ).…”

“…In this case, DNase I distributes between the aqueous media and the magnetic IL ( 86 ). Ion-tagged oligonucleotides with magnetic IL supports can be used for sequence-specific capture of DNA, which, in its turn, can be subsequently utilized as a template in PCR and real-time PCR and can be used for transformation of Escherichia coli ( 86 , 106 , 107 ). Similarly, poly-cytosine-tagged nucleotides and cobalt- or nickel-containing trihexyl(tetradecyl)phosphonium ILs can be used ( 108 ).…”

“…Magnetic ILs are employed as media for efficient extraction of DNA from water solutions, including cell lysates, and the extracts can be subsequently utilized as templates for PCR and real-time PCR ( 114–117 ). Ion-tagged oligonucleotides loaded on a magnetic IL support are successfully utilized for sequence-specific DNA extraction ( 106–108 , 118 , 119 ).…”

Ionic liquids: solvents and sorbents in sample preparation

“…Target DNA is then released from the ITO-MIL construct at elevated temperature and subjected to qPCR analysis. The [106] amount of DNA extracted using the MIL-ITO method was ultimately comparable to magnetic beads traditionally used for sequence-specific DNA extractions when applied to aqueous solutions and crude bacterial cell lysate. As shown in Fig.…”

“…The optimized reaction mixture consisting of magnesium chloride, EDTA, ovalbumin, and Tris (pH 8) mitigated PCR inhibition and pH changes caused by hydrolysis of the haloferrate anion. Very recently, Clark et al reported an approach based on a hydrophobic MIL support and ion-tagged oligonucleotides (ITO) that established the first particle-free alternative to magnet-based, targeted DNA extraction [106]. Figure 3A shows a schematic depiction of the MIL-ITO method.…”