Kinetics of multiplex hybridization: mechanisms and implications

Abstract

Quantitative analysis of DNA microarray data is complicated by uncertainties inherent to the experimental setup. Using computer simulations and real-time experimental results, we have previously demonstrated effects of multiplex reactions on a single sensing zone of an array, which may be a leading factor in erroneous interpretation of experimental data. We suggest here that a simplified three-component kinetic model may present a sufficient approximation to describe the general case of DNA sensing in a complex sample milieu. We show that, by analyzing the real-time hybridization kinetics of a nontarget species, we can perform quantitative analysis of unlabeled targets of interest within a broad dynamic range of concentrations.

Figure 1

Simulated hybridization curves for a multiplex hybridization with four components: target, competitor, and high and low background species. The composite curve (sum of all components) and effective background are also shown.

Figure 2

Hybridization curves representing the (A) target and (B) competitor kinetics as the number of background species increases from 0 to 5. Each progressive background species has a larger k_d value to simulate decreasing stability. All species are present at 5-nM concentrations.

Figure 3

Hybridization curves representing the (A and C) target and (B and D) competitor kinetics as the concentration of a high (A and B) or low (C and D) affinity background is changed from 0 nM to 10 nM. For the red curves the TOI was 1 nM and for the green curves the TOI was 5 nM. The other four background species have a constant concentration of 1 nM and the competitor concentration is 5 nM.

Figure 4

Hybridization curves; simulations represented by the black line (taken from previous figures) and fits represented by the dashed lines, representing the (A) target and (B) competitor kinetics as the concentration of different background species concentrations are changed while the competitor concentration is constant at 5 nM. For the red curves the target of interest was 1.0 nM and for the green curves the target of interest was 5 nM.

Figure 5

Hybridization curves (experimental data represented by dots while fits are represented by solid lines) representing the competitor kinetics as the concentration of deletion (A and C, with fixed 1 nM TMM) and TMM (B and D, with fixed 1 nM deletion) are shifted from 0 nM to 10 nM while the competitor concentration is constant at 5 nM. In panels C and D, the red curves are for target concentration of 1.6 nM and the green curves are for target concentration 5 nM; target is absent in panels A and B.