Diffusion Limitation: A Possible Source for the Occurrence of Doughnut Patterns on DNA Microarrays

Doughnut shaped hybridization patterns on DNA microarrays are mainly allocated to spotting or drying artifacts. The present study reports on results obtained from four different approaches that when combined generate a better view on the occurrence of these patterns. This study points out that doughnuts are not only formed during the spotting and drying process, but the hybridization process itself can be considered as an important cause. A combination of computer simulations, theoretical, optical, and experimental techniques shows how ring-shaped hybridization patterns occur when diffusion-limited conditions are present during the hybridization process. The theoretical assumptions as well as the simulations indicate that, for the basic geometry of a microarray hybridization experiment, a large amount of binding molecules reach the spot from the sides (and not from above the spot), leading to a preferential binding on the rims of the spot. These patterns seem to occur especially during hybridization with short oligonucleotides that have a very high binding probability and fast hybridization kinetics. Longer target DNA molecules lead to a more evenly distributed intensity signal. Furthermore, the diffusion-limited conditions also lead to pronounced hybridization intensity patterns on the scale of a whole spot block, where larger intensities are obtained on the edges of the block compared with the spots laying in the center of the block.