Nonequilibrium Hybridization Enables Discrimination of a Point Mutation within 5–40 °C

Abstract

Detection of point mutations and single nucleotide polymorphisms in DNA and RNA has a growing importance in biology, biotechnology, and medicine. For the application at hand, hybridization assays are often used. Traditionally, they differentiate point mutations only at elevated temperatures (>40 °C) and in narrow intervals (ΔT = 1–10 °C). The current study demonstrates that a specially designed multistranded DNA probe can differentiate point mutations in the range of 5–40 °C. This unprecedentedly broad ambient-temperature range is enabled by a controlled combination of (i) nonequilibrium hybridization conditions and (ii) a mismatch-induced increase of equilibration time in respect to that of a fully matched complex, which we dub “kinetic inversion”.

Thermodynamic framework to assess low abundance DNA mutation detection by hybridization

“…By using the concepts of DNA thermodynamics upon hybridization, the current technique has the advantage that mutations with low relative abundance can be detected in a higher parallelization level than PCR-based methods. In comparison to sequencing techniques, we can state that the process of hybridization is a very elementary reaction in which no enzymes are involved, which opens doors for further low-tech detection assays at physiological conditions [ 28 ]. Last but not least, the framework presented in this paper is platform-free and shows the advantage of a good description of the underlying physico-chemical principles.…”

Enhanced SNP-sensing using DNA-templated reactions through confined hybridization of minimal substrates (CHOMS)

“…12,13 The need to achieve more costeffective, faster analysis and higher single nucleotide resolution continues to drive technological developments. 14 Recent examples include amendments to existing technologies such as molecular beacons, 15,16 melting analysis, 17,18 environmentally sensitive uorescent nucleobases, [19][20][21][22][23][24] and strand displacement probes 25,26 or new technologies such as polymerase-amplied release of ATP (POLARA) 27 or graphene-based biosensors for real-time kinetic monitoring of hybridization. 28 The analysis of SNVs requires technologies with the highest nucleotide resolution to ascertain the polymorphism or variation.…”

DNA Three‐Way Junction for Differentiation of Single‐Nucleotide Polymorphisms with Fluorescent Copper Nanoparticles

“…With the development of chemical modification techniques, new strategies for SNPs detection have gradually emerged. Some are based on the labeling of DNA with fluorophores, organic molecules, or metal complexes, whereas some rely on the modification of certain spacer groups that connect the DNA to electrodes, nanochannels, or nanomaterials through chemical bonds, thereby exploiting fluorescent, electrical, or color signals . Although enzymes are avoided in these methods, the complicated process of chemical modification is tedious and expensive.…”