The design of microarrays is currently based on studies focusing on DNA hybridization reaction in bulk solution. However, the presence of a surface to which the probe strand is attached can make the solution-based approximations invalid, resulting in sub-optimum hybridization conditions. To determine the effect of surfaces on DNA duplex formation, the authors studied the dependence of DNA melting temperature (Tm) on target concentration. An automated system was developed to capture the melting profiles of a 25-mer perfect-match probetarget pair initially hybridized at 23 degrees C. Target concentrations ranged from 0.0165 to 15 nM with different probe amounts (0.030.82 pmol on a surface area of 1018 angstrom 2), a constant probe density (5 x 1012 molecules/cm2) and spacer length (15 dT). The authors found that Tm for duplexes anchored to a surface is lower than in-solution, and this difference increases with increasing target concentration. In a representative set, a target concentration increase from 0.5 to 15 nM with 0.82 pmol of probe on the surface resulted in a Tm decrease of 6 degrees C when compared with a 4 degrees C increase in solution. At very low target concentrations, a multi-melting process was observed in low temperature domains of the curves. This was attributed to the presence of truncated or mismatch probes. (c) 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012