Minisequencing on oligonucleotide microarrays: comparison of immobilisation chemistries

Abstract

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.

Detection of single nucleotide polymorphisms by minisequencing on a polypyrrole DNA chip designed for medical diagnosis

“…6 For solid-phasebased minisequencing reactions, several immobilization chemistries of oligonucleotides have been tested. 41 In particular, for glass supports, it has been demonstrated that the efficiency of genotyping by minisequencing is largely dependent on the type of oligonucleotide attachment. 41 In our solid-phase minisequencing assay, oligonucleotides are fixed on gold electrodes using pyrrole linkers in an electrochemical addressing process.…”

“…41 In particular, for glass supports, it has been demonstrated that the efficiency of genotyping by minisequencing is largely dependent on the type of oligonucleotide attachment. 41 In our solid-phase minisequencing assay, oligonucleotides are fixed on gold electrodes using pyrrole linkers in an electrochemical addressing process. 17,18 Combined with a real-time quality control performed at each stage of the production, the developed array, where each electrode corresponds to a different unit of hybridization, is characterized by a homogeneous probe density at the electrode surface with free 3 0 -OH groups available for enzymatic reactions.…”

Introduction to Microarray‐Based Detection Methods

“…Further important parameters to be considered during fabrication of the microarray are the probe concentration, spotting buffer and surface blocking strategies. Most of these features have been discussed thoroughly in the literature [79,98,172,200]. The most widely used microarray format is a planar glass slide (1 Â 3 in.).…”

“…Sequence analysis is performed by comparison of the hybridization patterns of the reference versus test sample. Such microarrays have been used for sequencing [74,197], detection of SNPs [74,98] and analysis of secondary structures [160]. Alternative detection methods have been suggested such as using a labeled common oligonucleotide primer that is extended to the site of the match or mismatch [106].…”

“…Various enzyme-assisted hybridization strategies (also used in SNP and resequencing assays [52,98]) are being applied because of their promise in strongly discriminating single mismatches located near the 3 0 -end of microarray probes [34,75,101,119,135,138,145].…”