title: Investigation of the multiple anchors approach in oligonucleotide microarray preparation using linear and stem-loop structured probes.
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.
authors.affiliation: Consiglio Nazionale delle Ricerche, Istituto di Tecnologie Biomediche, Universita' degli studi di Milano, Segrate, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Bordoni
authors.forename: Clarissa
authors.initials: C
authors.lastname: Consolandi
authors.forename: Bianca
authors.initials: B
authors.lastname: Castiglioni
authors.forename: Elena
authors.initials: E
authors.lastname: Busti
authors.forename: Luigi Rossi
authors.initials: LR
authors.lastname: Bernardi
authors.forename: Cristina
authors.initials: C
authors.lastname: Battaglia
authors.forename: Gianluca
authors.initials: G
authors.lastname: De Bellis
mesh_terms: D001483:Base Sequence; D009690:Nucleic Acid Conformation; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes / Q000737:chemistry*; D013873:Thionucleotides / Q000737:chemistry
chemical_list: D015345:Oligonucleotide Probes; D013873:Thionucleotides