Minisequencing on oligonucleotide microarrays: comparison of immobilisation chemistries.
In the microarray format of the minisequencing method multiple oligonucleotide primers immobilised on a glass surface are extended with fluorescent ddNTPs using a DNA polymerase. The method is a promising tool for large-scale single nucleotide polymorphism (SNP) detection. We have compared eight chemical methods for covalent immobilisation of the oligonucleotide primers on glass surfaces. We included both commercially available, activated slides and slides that were modified by ourselves. In the comparison the differently derivatised glass slides were evaluated with respect to background fluorescence, efficiency of attaching oligonucleotides and performance of the primer arrays in minisequencing reactions. We found that there are significant differences in background fluorescence levels among the different coatings, and that the attachment efficiency, which was measured indirectly using extension by terminal transferase, varied largely depending on which immobilisation strategy was used. We also found that the attachment chemistry affects the genotyping accuracy, when minisequencing on microarrays is used as the genotyping method. The best genotyping results were observed using mercaptosilane-coated slides attaching disulfide-modified oligonucleotides.
Lindroos
K
K
Molecular Medicine, Department of Medical Sciences, Uppsala University, 75185 Uppsala, Sweden.
Liljedahl
U
U
Raitio
M
M
Syvänen
A C
AC
D004252:DNA Mutational Analysis / Q000379:methods; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D005453:Fluorescence; D005838:Genotype; D005898:Glass; D006801:Humans; D017879:Isothiocyanates / Q000378:metabolism; D007834:Lasers; D009154:Mutation / Q000235:genetics; D019001:Nitric Oxide Synthase / Q000235:genetics; D052250:Nitric Oxide Synthase Type III; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D012821:Silanes / Q000378:metabolism
D017931:DNA Primers; D004220:Disulfides; D017879:Isothiocyanates; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin; D012821:Silanes; C037152:isothiocyanic acid; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D004253:DNA Nucleotidylexotransferase