Statistical Thermodynamics and Kinetics of DNA Multiplex Hybridization Reactions

Abstract

A general analytical description of the equilibrium and reaction kinetics of DNA multiplex hybridization has been developed. In this approach, multiplex hybridization is considered to be a competitive multichannel reaction process: a system wherein many species can react both specifically and nonspecifically with one another. General equations are presented that can consider equilibrium and kinetic models of multiplex hybridization systems comprised, in principle, of any number of targets and probes. Numerical solutions to these systems for both equilibrium and kinetic behaviors are provided. Practical examples demonstrate clear differences between results obtained from more common simplex methods, in which individual hybridization reactions are considered to occur in isolation; and multiplex hybridization, where desired and competitive cross-hybrid reactions between all possible pairs of strands are considered. In addition, sensitivities of the hybridization process of the perfect match duplex, to temperature, target concentration, and existence of sequence homology with other strands, are examined. This general approach also considers explicit sequence-dependent interactions between targets and probes involved in the reactions. Sequence-dependent stabilities of all perfect match and mismatch duplex complexes are explicitly considered and effects of relative stability of cross-hybrid complexes are also explored. Results reveal several interdependent factors that strongly influence DNA multiplex hybridization behavior. These include: relative concentrations of all probes and targets; relative thermodynamic stability of all perfect match and mismatch complexes; sensitivity to temperature, particularly for mismatches; and amount of sequence homology shared by the probe and target strands in the multiplex mix.

Influence of Buffer Species on the Thermodynamics of Short DNA Duplex Melting: Sodium Phosphate versus Sodium Cacodylate

“…Although errors apply uniformly to all reacting species in the reaction (i.e., perfect match duplexes or cross-hybrids), quantitative and accurate predictions of hybridization behavior requires explicit knowledge of the sources of thermodynamic stability. 10 Conclusions DSC melting studies of 53 short DNA duplexes in ∼1.0 M Na + solvent containing either sodium cacodylate or sodium phosphate buffer revealed statistically significant differences in stability associated with the different buffer species. Higher stability was observed in the NaCl/phosphate than in the NaCl/ cacodylate buffer.…”

Biophysical properties of nucleic acids at surfaces relevant to microarray performance

“…However, “real” microarrays operate in “multiplex” mode, where many other ssDNA strands and their possible duplex pairings, even as mismatches with finite metastability, are present and competitively operative. [211, 214] Few studies report deviations for perfect match probabilities or kinetics in the presence of strand multiplex bias where different ssDNA concentrations and relative ranges of strand-strand affinities for a multitude of various duplex reactions compete simultaneously, either in bulk or at surfaces. Several additional essential features of multiplex hybridization must be considered.…”

Real-time DNA microarrays: reality check

“…Improved probe design usually is based on sequence text analysis and on nearest-neighbour thermodynamic calculations [42]. A more advanced version of probe design is based on finding the unique thermodynamic signature of the probe–target hybrids based on statistical thermodynamics calculations [8]. It is aimed at minimizing cross-hybridization with homologous sequences (i.e.…”

“…[4,5]. The fundamental problem we have to deal with when conducting microarray experiments is the multiplex environment of the surface capture [6–8]. Indeed, multiple DNA (RNA) targets interact with each sensing zone (spot) on the surface, and each target can potentially partition among multiple spots.…”