Abstract
| Original language | English |
|---|---|
| Journal | Analytical Biochemistry |
| Volume | 352 |
| Issue number | 2 |
| Pages (from-to) | 188-197 |
| ISSN | 0003-2697 |
| DOIs | |
| Publication status | Published - 2006 |
Cite this
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Detection of mutations using microarrays of poly(C)10-poly(T)10 modified DNA probes immobilized on agarose films. / Dufva, Hans Martin; Petersen, Jesper; Stoltenborg, M.; Birgens, H.; Christensen, C.B.V.
In: Analytical Biochemistry, Vol. 352, No. 2, 2006, p. 188-197.Research output: Contribution to journal › Journal article › Research › peer-review
TY - JOUR
T1 - Detection of mutations using microarrays of poly(C)10-poly(T)10 modified DNA probes immobilized on agarose films
AU - Dufva, Hans Martin
AU - Petersen, Jesper
AU - Stoltenborg, M.
AU - Birgens, H.
AU - Christensen, C.B.V.
PY - 2006
Y1 - 2006
N2 - 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.
AB - 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.
U2 - 10.1016/j.ab.2006.03.008
DO - 10.1016/j.ab.2006.03.008
M3 - Journal article
C2 - 16615930
VL - 352
SP - 188
EP - 197
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 2
ER -