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

Dye-related Factors
equilibrium, an underlying assumption for interpreting a Microarray experiment [5,
10]. Achieving equilibrium is a mandate of DNA Microarray experiments in order to
ensure a reliable, reproducible, measurement of transcript concentration [5, 10].
After the chip has been incubated with the labeled target/solvent mixture, the unbound reagents
are washed away, and the chip is assayed for the remaining presence of the label, which in most
cases is a fluorescent dye [13, 14, 16, 38, 39]. The dye absorbs specific wavelength frequencies
and emits photons at a second frequency which are captured and amplified by a CCD camera or
similar device [40-42]. The readout for the measurement is an image, either from a scanner or
actual imager, with varying intensities in ‘fluorescent units’ at specific locations [14, 15].
Extraction of the intensity at the position known to coincide with a probe is used to infer the type
and amount of target present in the duplex. Hence, reliable assessment of a Microarray
experiment is dependent on knowing whether the reaction has reached equilibration and how
many fluorescent units are emitted per incorporated dye and the number of such dye molecules
per target. Dyes may be incorporated at internal positions with modified nucleotides, or may be
incorporated onto the 5’ end of the target strand through catalytic hydrolysis of the phosphate tail
[5]. In either case the enzymes used have preferential incorporation rates with different dyes [40-
42]. A common multi-label strategy is co-hybridization, in which a control and experimental
sample are labeled with distinct dyes, mixed and hybridized to the same array, with emission
filters being used to separate the signals [14, 15]. These two-dye experiments need to include the
distinctive properties of the chosen dyes in the experimental design, so as to avoid quenching and
dye quantum yield bias in the final comparisons [38, 39]. Affymetrix protocols do not use co-
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