Sample A: Cover Page of Thesis, Project, or Dissertation Proposal

Microarrays: Hybridization and Signal
DNA Microarray assays are based upon the principal of complementary nucleotides binding in
solution to form a stable anti-parallel duplex [10, 21]. Hydrogen bonds dictate the kinetics of the
nucleotide pairings, and base-stacking interactions govern the stability of the resultant product
[22-24]. The factors affecting the rate and extent of these endothermic reactions have been
extensively studied, including the ionic conditions, temperature and solvent characteristics under
which the single strands will form hybrids [22, 24-27]. However an important and not well
understood distinction in a Microarray experiment is that the probes are fixed to a surface and
may not freely diffuse [10, 14]; in addition the reactions are not composed of single sets of
reactants but a highly multiplex set of unimolecular and bimolecular reactions within and
between probes and targets.
That is, since a heterogeneous mixture of transcript targets exists, duplex reactions compete to
form stable compounds, some containing a variety of mismatches that dissociate slowly [28], and
therefore, to optimize the measurement of the best matches, the mixture of reactions must be
allowed to reach a state of equilibrium [3, 10]. The formation of sub-optimal duplexes is termed
cross-hybridization, and those targets with high enough sequence similarity to compete for stable
products are ‘false positive’ signals under the usual guidelines of interpretation [17, 20]. The
combination of desired and undesired hybridizations generates a fluorescent signal, recorded as a
spot of high intensity that is extracted by the instrument’s scanning software. The amount and
position of dye incorporated into the target will clearly also affect the intensity of the signal and
must be accounted for in quantitative interpretations [5].
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