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Nucleic Acids Research Advance Access published online on September 19, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn600
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

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

Lena Poulsen1, Martin Jensen Søe2, Detlef Snakenborg1, Lisbeth Birk Møller3 and Martin Dufva1,*

1DTU Nanotech, Department of Micro and Nanotechnology, Technical University of Denmark, Oersteds Plads, Bld. 345 East, DK-2800 Lyngby, 2Bioneer A/S, Kogle Allé 2, DK-2970 Hørsholm and 3Kennedy Centre, Gamle Landevej 7, DK-2600 Glostrup, Denmark

*To whom correspondence should be addressed. Tel: +45 4525 6324; Fax: +45 4588 7762; Email: martin.dufva{at}nanotech.dtu.dk

Received June 26, 2008. Revised September 4, 2008. Accepted September 4, 2008.

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 gene-specific sequence in discrete steps along the 60-mer probes. The study was performed using Agilent 8 x 15 000 probes custom-made arrays and a home-built 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 4 x SSC, while probes placed away from the surface required 0.35 x SSC 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.


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