Multi-stringency wash of partially hybridized 60-mer probes reveals that the stringency along the probe decreases with distance from the microarray surface

Here, we describe a multi-parametric study of DNA
hybridization to probes with 20–70% G+C content.
Probes were designed towards 71 different sites/
mutations in the phenylalanine hydroxylase gene.
Seven probe lengths, three spacer lengths and six
stringencies were systematically varied. The three
spacer lengths were obtained by placing the genespecific
sequence in discrete steps along the 60-mer
probes. The study was performed using Agilent
8315 000 probes custom-made arrays and a homebuilt
array washer providing different stringencies
to each of the eight sub-arrays on the slides.
Investigation of hybridization signals, specificity
and dissociation curves indicated that probes
close to the surface were influenced by an additional
stringency provided by the microarray surface.
Consistent with this, probes close to the surface
required 43SSC, while probes placed away from
the surface required 0.353SSC wash buffers in
order to give accurate genotyping results. Multiple
step dissociation was frequently observed for
probes placed furthest away from surface, but not
for probes placed proximal to the surface, which is
consistent with the hypothesis that there is different
stringency along the 60-mer. The results have impact
on design of probes for genotyping, gene expression
and comparative genome hybridization analysis.