Emerging technologies for hybridization based single nucleotide polymorphism detection

Abstract

Detection of single nucleotide polymorphisms (SNPs) is a crucial challenge in the development of a novel generation of diagnostic tools. Accurate detection of SNPs can prove elusive, as the impact of a single variable nucleotide on the properties of a target sequence is limited, even if this sequence consists of only a few nucleotides. New, accurate and facile strategies for the detection of point mutations are therefore absolutely necessary for the increased adoption of point-of-care molecular diagnostics. Currently, PCR and sequencing are mostly applied for diagnosing SNPs. However these methods have serious drawbacks as routine diagnostic tools because of their labour intensity and cost. Several new, more suitable methods can be applied to enable sensitive detection of mutations based on specially designed hybridization probes, mutation recognizing enzymes and thermal denaturation. Here, an overview is presented of the most recent advances in the field of fast and sensitive SNP detection assays with strong potential for integration in point-of-care tests.

Single-Molecule Counting of Point Mutations by Transient DNA Binding

“…Current efforts have focused on enhancing single-nucleotide selectivity, including the development of digital PCR3, barcode-based assays4, nanopore approaches5 and next-generation sequencing6. Hybridization probes7 (such as molecular beacons, binary probes, and artificial modified probes) effectively detect mutations in DNA sequences where the corresponding wild type and mutant alleles are known. The specificity of these probes is dependent on their hybridization thermodynamics, rendering it typically poor at room temperature.…”

An innovative application of time-domain spectroscopy on localized surface plasmon resonance sensing

“…The sequences of the DNA strands used in this work, including two biotinylated probes and one target strand, were shown in Table S1 (Supplementary Information). The individual probes binding to a relatively short fragment of the target, makes the short duplexes extremely sensitive to single nucleotide substitutions262728. When the short duplexes forming, gold nanoparticles aggregated, and a large shift in the spectral centroid of the LSPR extinction spectrum was caused only in the presence of the fully complementary targets, resulting in a remarkable increase in both the specificity and sensitivity.…”

The Osmium Tetroxide Bipyridine‐labeled DNA Probe: Hairpin Conformations and Characterization of Redox‐label Behavior

“…As compared to either linear hybridization with a labeled target , or an end‐labeled probe with no stem‐loop structure , hybridization is more difficult to achieve energetically in the MB, as it involves a strand‐displacement of the self‐complementary duplex for the target. Thus small differences in stabilization energy of the final duplex, such as those caused by single‐base mismatches, are amplified by a high reaction barrier, and can be detected more easily than in a simple labeled‐target linear hybridization assay ; in this case, destabilization of the duplex at the electrode caused by the single‐base mismatch is insufficient to be clearly recognized by signal differences .…”