Extraction and Purification of DNA from Complex Biological Sample Matrices Using Solid-Phase Microextraction Coupled with Real-Time PCR

The determination of extremely small quantities of DNA from complex biological sample matrices represents a significant bottleneck in nucleic acid analysis. In this study, polymeric ionic liquid (PIL)-based solid-phase microextraction (SPME) was applied for the extraction and purification of DNA from crude bacterial cell lysate with subsequent quantification by real-time PCR (qPCR) analysis. Using an on-fiber ultraviolet initiated polymerization technique, eight different PIL sorbent coatings were generated and their DNA extraction performance evaluated using qPCR. The PIL sorbent coating featuring halide anions and carboxylic acid groups in the cationic portion exhibited superior DNA extraction capabilities when compared to the other studied PILs and a commercial polyacrylate SPME fiber. Electrostatic interactions as well as an ion-exchange mechanism were identified as the driving forces in DNA extraction by the PIL sorbents. The selectivity of the PIL sorbent coating for DNA was demonstrated in the presence of PCR inhibitors at high concentration, where a quantifiable amount of template DNA was extracted from aqueous samples containing CaCl2 and FeCl3. Furthermore, the PIL-based SPME method was successfully applied for the extraction of DNA from crude bacterial cell lysate spiked with 1 pg mL−1 template DNA without requiring the use of organic solvents or centrifugation steps. Following PIL-based SPME of DNA from a dilute cell lysate, the qPCR amplification efficiency was determined to be 100.3%, demonstrating the feasibility of the developed method to extract high purity DNA from complex sample matrices

Maximizing Ion-tagged Oligonucleotide Loading on Magnetic Ionic Liquid Supports for the Sequence-Specific Extraction of Nucleic Acids

Targeted nucleic acid analysis requires the highly selective extraction of desired DNA fragments in order to minimize interferences from samples with abundant heterogeneous sequences. We previously reported a method based on functionalized oligonucleotide probes known as ion-tagged oligonucleotides (ITOs) that hybridize with complementary DNA targets for subsequent capture using a hydrophobic magnetic ionic liquid (MIL) support. Although the ITO-MIL approach enriched specific DNA sequences in quantities comparable to a commercial magnetic bead-based method, the modest affinity of the ITO for the hydrophobic MIL limited the yield of DNA targets, particularly when stringent wash conditions were applied to remove untargeted DNA. Here, we report the synthesis and characterization of a series of ITOs in which functional groups were installed within the cation and anion components of the tag moiety in order to facilitate loading of the ITO to the MIL support phase. In addition to hydrophobic interactions, we demonstrate that π-π stacking and fluorophilic interactions can be exploited for loading oligonucleotide probes onto MILs. Using a disubstituted ion-tagged oligonucleotide (DTO) possessing two linear C8 groups, nearly quantitative loading of the probe onto the MIL support was achieved. The enhanced stability of the DTO within the MIL solvent permitted successive wash steps without the loss of the DNA target compared to a monosubstituted ITO with a single C8 group that was susceptible to increased loss of analyte. Furthermore the successful capture of a 120 bp KRAS fragment from human plasma samples followed by real-time quantitative polymerase chain reaction (qPCR) amplification is demonstrated

Advances in Mutation Detection Using Loop-Mediated Isothermal Amplification

Detection of mutations and single-nucleotide polymorphisms is highly important for diagnostic applications. Loop-mediated isothermal amplification (LAMP) is a powerful technique for the rapid and sensitive detection of nucleic acids. However, LAMP traditionally does not possess the ability to resolve single-nucleotide differences within the target sequence. Because of its speed and isothermal nature, LAMP is ideally suited for point-of-care applications in resource-limited settings. Recently, different approaches have been developed and applied to enable single-nucleotide differentiation within target sequences. This Mini-Review highlights advancements in mutation detection using LAMP. Methods involving primer design and modification to enable sequence differentiation are discussed. In addition, the development of probe-based detection methods for mutation detection are also covered