Investigation of the multiple anchors approach in oligonucleotide microarray preparation using linear and stem-loop structured probes.

Roberta Bordoni, Clarissa Consolandi, Bianca Castiglioni, Elena Busti, Luigi Rossi Bernardi, Cristina Battaglia, Gianluca De Bellis

Research output: Contribution to journalArticle

Abstract

Enzyme-mediated reactions are a useful tool in mutation detection when using a microarray format. Discriminating probes attached to the surface of a DNA chip have to be accessible to target DNA and to the enzyme (ligase or polymerase) that catalyses the formation of a new phosphodiester bond. This requires an appropriate chemical platform. Recently, an oligonucleotide hairpin architecture incorporating multiple phosphorothioate moieties along the loop has been proposed as an effective approach to solid-phase minisequencing. We have explored in depth several variables (stem length, number of phosphorothioates, stem-loop architecture versus linear structure) involved in this strategy by using a solid-phase ligation reaction. Microarrays were fabricated either from aminosilyl-modified glass or from aminated polymeric surfaces made of poly-lysine. Both platforms were bromoacetylated and reacted with thiophosphorylated oligonucleotides. The resulting microarrays were tested using either a synthetic template or a PCR-amplified 16S rRNA genomic region as the target sequence. Our results confirm the robustness of the proposed chemistry. We extend its range of application to solid-phase ligation, demonstrating the effectiveness of multiple anchors and suggest that linear oligonucleotides incorporating multiple phosphorothioates are equivalent to their hairpin-structured counterparts.

Original languageEnglish
JournalNucleic Acids Research
Volume30
Issue number8
Publication statusPublished - Apr 15 2002

ASJC Scopus subject areas

  • Genetics

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