Detection of mutations using microarrays of poly(C)10-poly(T)10 modified DNA probes immobilized on agarose films

Hans Martin Dufva, Jesper Petersen, M. Stoltenborg, H. Birgens, C.B.V. Christensen

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    Allele-specific hybridization to a DNA microarray call be a useful method for genotyping patient DNA. In this article, we demonstrate that 13- to 17-base oligonucleotides tagged with a poly(T)10-poly(C)10 tail (TC tag), but otherwise unmodified, can be crosslinked by UV light irradiation to an agarose film grafted onto unmodified glass. Microarrays of TC-tagged probes immobilized on the agarose film can be used to diagnose Mutations in the human P-globin gene, which encodes the beta-chains in hemoglobin. Although the probes differed widely regarding inciting point temperature (similar to 20 degrees C), a single stringency wash still gave sufficiently high discrimination signals between perfect match and mismatch probes to allow robust mutation detection. In all, 270 genotypings were performed on patient materials, and no genotype was incorrectly classified. Quality control experiments conducted using a target DNA specific for the TC tag of the immobilized probes showed that the spotting and hybridization procedure had a variance of 20%, indicating that signal differences as low as twofold could be detected between perfect match and mismatch. Together, our results show that the use of inicroarrays of TC-tagged probes that have been immobilized on agarose films grafted onto glass is a robust and inexpensive genotyping method. (c) 2006 Elsevier Inc. All rights reserved.
    Original languageEnglish
    JournalAnalytical Biochemistry
    Volume352
    Issue number2
    Pages (from-to)188-197
    ISSN0003-2697
    DOIs
    Publication statusPublished - 2006

    Cite this