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 (fa) 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 characterizing the efficiency of SNP discrimination is the limit selectivity of hybridization, which determines the utmost value of fa 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 fa 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 fa has its maximum value. The dependency of the fa 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 oli- gonucleotides or their derivatives.
Highlights
The method of molecular hybridization of oligonucleotide probes with nucleic acids in order to reveal specific sequences has been widely utilized in molecular biology [1,2]
It is necessary to use “selectivity” as the obligatory term if we consider the quantitative parameters characterizing the ability of an oligonucleotide probe to distinguish one sequence from the other one
We considered the variant implying that the analyzed site was unique, i.e., occurred once in both native and mutated form of the analyzed target
Summary
The method of molecular hybridization of oligonucleotide probes with nucleic acids in order to reveal specific sequences has been widely utilized in molecular biology [1,2]. There are several strategies for enhancing selectivity of hybridization between oligonucleotides and nucleic acids (NA), including variation of hybridization conditions (temperature, the probe concentration, and the buffer composition) [7,8,9] and competitive inhibition through the use of stringency clamping [10] or molecular beacons [11,12]. Changing the probe length [17] or using tandem short probes [18,19,20,21] has been shown to influence selectivity Another strategy involves the use of nanoparticles bearing immobilized oligonucleotide probes [22,23,24]. Modifications can be conventionally divided in two groups: (1) those that increase the thermostability of the DNA-probe complex (PNA [25]; LNA [4]; cyclic, cross-linked, and bicyclic oligo-nucleotides [26,27]; 3’-minor groove binder-DNA probes [28]; HNA and ANA [29], etc.) and (2) those that decrease the thermostability of the DNA-probe complex due to an artificial mismatch [30,31], a non-nucleotide insert [32,33] and others
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