Analytical consideration of the selectivity of oligonucleotide hybridization

Abstract

Systematic analysis of factors determining efficiency in discrimination of a point substitution (SNP) within specific DNA sequences was carried out in the context of hybridization approach. There are two types of selectivity that are critical for the rational design of highly specific oligonucleotides probes. The first type is the real selectivity of hybridization (f  ) that is the ratio of association degrees of targets with an oligonucleotide probe upon the perfect and imperfect complex formation. This type of selectivity reflects the level of discrimination between matched and mismatched signals, which is determined both by experimental conditions and the thermodynamics of oligonucleotide hybridization. The second parameter characterizeing the efficiency of SNP discrimination is the limit selectivity of hybridization, which determines the utmost value of f  at a given temperature. This value can be calculated as the ratio of corresponding equilibrium association constants of perfect and imperfect complex formation determined purely by thermodynamics. We have shown that the f  function is the most reliable characteristic describing the hybridization selectivity. For the analytical system designed to reveal any type of perturbation in DNA (e.g. SNP or modification), there is usually a temperature at which f  has its maximum value. The dependency of the f  maximum on different experimental parameters as well as the structural characteristics of a probe are described in details. The results allowed us to postulate points of principle to rationally design the most selective probes on the basis of oligonucleotides or their derivatives.

Rapid KRAS Mutation Detection via Hybridization-Induced Aggregation of Microbeads

“…In principle, a HIA-based method for mutation detection (HIA-MD) relies on differences in the thermal stabilities of perfectly matched and mismatched target-probe pairs to achieve allelic discrimination. Despite extensive knowledge about nucleic acid hybridization, meticulous empirical evaluation and assay-specific heuristics are required to establish the conditions that will ultimately permit two sequences differing by a single base to be distinguished . Key parameters affect the thermodynamics of duplex formation and, therefore, were acutely considered in the development and optimization of this assay.…”

“…Despite extensive knowledge about nucleic acid hybridization, meticulous empirical evaluation and assayspecific heuristics are required to establish the conditions that will ultimately permit two sequences differing by a single base to be distinguished. 28 Key parameters affect the thermodynamics of duplex formation and, therefore, were acutely considered in the development and optimization of this assay. These included probe design, buffer composition, and target parameters.…”

Mapping and sequencing DNA using nanopores and nanodetectors

“…The use of longer probes resolves the repeats but the size of required probe libraries grow much faster than the reconstructable length. Furthermore, selectivity with respect to mismatches also suffers as probe length increases . Thus, the probe lengths that are both reasonable with respect to numbers and selective with respect to mismatches limits the size of the DNA that can be sequenced to only hundreds of base pairs.…”