  HDONA technology is predicated on two ideas. First, the differential between
high-affinity (perfect match, PM) and lower-affinity (mismatch, MM) probes is
used to minimize cross-hybridization. Second, several short probes along the
transcript are combined, introducing redundancy. Both ideas have shown problems
in practice: MMs are often brighter than PMs, and it is hard to combine the
pairs because their brightness often spans decades. Previous analysis suggested
these problems were sequence-related; publication of the probe sequences has
permitted us an in-depth study of this issue. Our results suggest that
fluorescently labeling the nucleotides interferes with mRNA binding, causing a
catch-22 since, to be detected, the target mRNA must both glow and stick to its
probe: without labels it cannot be seen even if bound, while with too many it
won't bind. We show that this conflict causes much of the complexity of HDONA
raw data, suggesting that an accurate physical understanding of hybridization
by incorporating sequence information is necessary to perfect microarray
analysis.
