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Nucleic Acids Research 2005 33(3):e33; doi:10.1093/nar/gni034
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Published online 18 February 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org

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Comparisons of substitution, insertion and deletion probes for resequencing and mutational analysis using oligonucleotide microarrays

Mazen W. Karaman, Susan Groshen1, Chi-Chiang Lee, Brian L. Pike and Joseph G. Hacia*

The Institute for Genetic Medicine, University of Southern California Los Angeles, CA 90089, USA 1 Department of Preventive Medicine, Norris Comprehensive Cancer Center, University of Southern California Los Angeles, CA 90089, USA

*To whom correspondence should be addressed at The Institute for Genetic Medicine, University of Southern California, 2250 Alcazar Street, IGM 240 Los Angeles, CA 90089, USA. Tel: +1 323 442 3030; Fax: +1 323 442 2764; Email: hacia{at}usc.edu

Received December 2, 2004. Revised January 20, 2005. Accepted February 4, 2005.

Although oligonucleotide probes complementary to single nucleotide substitutions are commonly used in microarray-based screens for genetic variation, little is known about the hybridization properties of probes complementary to small insertions and deletions. It is necessary to define the hybridization properties of these latter probes in order to improve the specificity and sensitivity of oligonucleotide microarray-based mutational analysis of disease-related genes. Here, we compare and contrast the hybridization properties of oligonucleotide microarrays consisting of 25mer probes complementary to all possible single nucleotide substitutions and insertions, and one and two base deletions in the 9168 bp coding region of the ATM (ataxia telangiectasia mutated) gene. Over 68 different dye-labeled single-stranded nucleic acid targets representing all ATM coding exons were applied to these microarrays. We assess hybridization specificity by comparing the relative hybridization signals from probes perfectly matched to ATM sequences to those containing mismatches. Probes complementary to two base substitutions displayed the highest average specificity followed by those complementary to single base substitutions, single base deletions and single base insertions. In all the cases, hybridization specificity was strongly influenced by sequence context and possible intra- and intermolecular probe and/or target structure. Furthermore, single nucleotide substitution probes displayed the most consistent hybridization specificity data followed by single base deletions, two base deletions and single nucleotide insertions. Overall, these studies provide valuable empirical data that can be used to more accurately model the hybridization properties of insertion and deletion probes and improve the design and interpretation of oligonucleotide microarray-based resequencing and mutational analysis.


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