Kinetics of oligonucleotide hybridization to photolithographically patterned DNA arrays.

The hybridization kinetics of oligonucleotide targets to oligonucleotide probe arrays synthesized using photolithographic fabrication methods developed by Affymetrix have been measured. Values for the fundamental adsorption parameters, k(a), k(d), and K, were determined at both room temperature and 45 degrees C by monitoring the hybridization of fluorescently labeled targets to the array. The values for these parameters and the adsorbed target density (<or=1 pmol/cm(2) at saturation) agree relatively well with published values for arrays fabricated by immobilizing intact probes. The isotherms can be fit well with the Sips model, a generalization of the Langmuir model that allows for multiple binding energies. However, binding to these arrays also displays certain characteristics that may result from the close spacing of probes on the array. At high target concentrations and 22 degrees C, an "overshoot" is observed, wherein a large amount of target binds rapidly and then desorbs to a final plateau. This binding mode may be a result of the unique nature of photolithographically patterned arrays given that targets initially can bind in lower stability binding modes by partially adsorbing to a probe and its neighbor(s), a process that can be viewed as a form of competitive hybridization for overlapping sites on a given probe. Using the values measured for k(a) and k(d), an analytical model that accounts for this behavior is proposed. Alternatively, at 45 degrees C, the adsorption approaches an initial plateau, rather than an overshoot, and then undergoes a "secondary rise" to a final value. A potential explanation for this phenomenon that is compatible with the analysis at lower temperature is offered.