Toward Non-Enzymatic Ultrasensitive Identification of Single Nucleotide Polymorphisms by Optical Methods

Abstract

Single nucleotide polymorphisms (SNPs) are single nucleotide variations which comprise the most wide spread source of genetic diversity in the genome. Currently, SNPs serve as markers for genetic predispositions, clinically evident disorders and diverse drug responses. Present SNP diagnostics are primarily based on enzymatic reactions in different formats including sequencing, polymerase-chain reaction (PCR) and microarrays. In these assays, the enzymes are applied to address the required sensitivity and specificity when detecting SNP. On the other hand, the development of enzyme-free, simple and robust SNP sensing methods is in a constant focus in research and industry as such assays allow rapid and reproducible SNP diagnostics without the need for expensive equipment and reagents. An ideal method for detection of SNP would entail mixing a DNA or RNA target with a probe to directly obtain a signal. Current assays are still not fulfilling these requirements, although remarkable progress has been achieved in recent years. In this review, current SNP sensing approaches are described with a main focus on recently introduced direct, enzyme-free and ultrasensitive SNP sensing by optical methods.

Novel Signal-Enhancing Approaches for Optical Detection of Nucleic Acids—Going beyond Target Amplification

“…According to recent research, probe specificity can be dramatically improved using synthetic nucleic acid analogues such as locked nucleic acids oxo-LNA (L) and functionalized 2'-amino-LNA analogues, Figure 3A; [4]. The structural and thermodynamic parameters of LNA/DNA oligonucleotides are different compared to non-modified DNA.…”

“…Moreover, LNA molecules can be combined with fluorescent markers in the form of either fluorogenic probes or dyes that can be directly attached to the 2′-amino position of 2′-amino-LNA ( Figure 3). Short fluorescent LNA/DNA probes containing bright photostable polyaromatic hydrocarbons (PAHs) such as pyrene, (phenylethynyl)pyrenes and perylene represent a class of probes with the advantage of high target specificity and sensitivity [4]. Without target amplification, genotyping of HIV-1 protease encoding gene fragment (HIV PR) was performed with a pyrene probe yielding a LOD below 5 nM [24].…”

“…Complexes between DNA and negatively charged gold nanoparticles (Au-nps) have been studied for many years and have recently been found useful in specific nucleic acid detection with colorimetry [4,38]. These assays utilize the dispersion and aggregation of the Au-nps, either with a cross-linking or non-cross-linking aggregation mechanism [48].…”

“…In a recent paper we gave an overview of enzymatic strategies involving amplification of nucleic acid targets in comparison to rapidly developing enzyme-free assays [4]. As discussed in this paper, PCR-independent methods without additional signal enhancing strategies yielded limit of target detection (LOD) values of 5 nM, which is not suitable for most biological applications [3,4]. Signal-enhancing strategies are therefore envisioned as a necessary tool for reliable, sensitive detection in enzyme-free assays.…”

Fluorescent Nucleic Acid Analogues in Research and Clinical Diagnostics

“…Current methods for DNA/RNA diagnostics can be divided in two large categories of assays: (1) enzymatic high-throughput assays and (2) enzyme-free assays that are portable and less expensive (Khan 2014;Lyubchenko et al 2011;Astakhova 2014;Turner 2013). The former methods typically apply enzymes and state-of-the-art equipment in order to achieve a necessary degree of sensitivity and specificity of diagnostics, e.g., sequencing, polymerase-chain reaction PCR, and microarrays.…”