Detection of mutations using microarrays of poly(C)10-poly(T)10 modified DNA probes immobilized on agarose films.
Allele-specific hybridization to a DNA microarray can 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 beta-globin gene, which encodes the beta-chains in hemoglobin. Although the probes differed widely regarding melting point temperature ( approximately 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 microarrays of TC-tagged probes that have been immobilized on agarose films grafted onto glass is a robust and inexpensive genotyping method.
Dufva
Martin
M
Microarray Group, Department of Micro and Nanotechnology, Technical University of Denmark, DK-2800 Kongens Lyngby, Denmark. mdu@mic.dtu.dk
Petersen
Jesper
J
Stoltenborg
Michael
M
Birgens
Henrik
H
Christensen
Claus B V
CB
D000483:Alleles; D001483:Base Sequence; D004247:DNA / Q000032:analysis / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods; D015342:DNA Probes / Q000235:genetics* / Q000528:radiation effects; D005838:Genotype; D005914:Globins / Q000235:genetics; D006801:Humans; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D009841:Oligonucleotides / Q000235:genetics; D011066:Poly C / Q000235:genetics* / Q000528:radiation effects; D011071:Poly T / Q000235:genetics* / Q000528:radiation effects; D012685:Sepharose / Q000737:chemistry*; D014466:Ultraviolet Rays
D015342:DNA Probes; D008567:Membranes, Artificial; D009841:Oligonucleotides; D011071:Poly T; D011066:Poly C; D005914:Globins; D004247:DNA; D012685:Sepharose