Competitive hybridization kinetics reveals unexpected behavior patterns.

Although the kinetics of hybridization between a soluble polynucleotide and an immobilized complementary sequence have been studied by others, it is almost universally assumed that the interaction between each probe/target pair can be treated as a separate event. This simplifies the mathematics considerably, but it can give a false picture of the extent of hybridization that one achieves at equilibrium as well as the relative quantities of each hybridized pair during the approach to equilibrium. Here we solve the relevant kinetics equations simultaneously using Mathematica as a simulation language. Among the interesting results of this study are that, for certain circumstances, the relative ratio of incorrect to correct hybrids can change dramatically with time; that the relative abundances of two pairs are not what one would expect based on their equilibrium dissociation constants; that the volume of a wash solution after hybridization can have a large effect on results; and the fact that a short wash is typically better than a long one. We show that an optimum wash time exists for a given set of conditions. In addition, the ratio of soluble to insoluble (spotted) molecules can influence results substantially. Finally, the true levels of rare transcripts can be masked by the presence of highly abundant ones. Code is supplied to enable others to study conditions beyond those presented in this article.