Nucleic Acids Research, 2002, Vol. 30, No. 8 e34
© 2002 Oxford University Press
Investigation of the multiple anchors approach in oligonucleotide microarray preparation using linear and stem–loop structured probes
Consiglio Nazionale delle Ricerche, Istituto di Tecnologie Biomediche and 1Universita’ degli studi di Milano, Dipartimento di Scienze e Tecnologie Biomediche and CISI, LITA, Via Fratelli Cervi 93, 20090 Segrate, Italy
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.
* To whom correspondence should be addressed. Tel: +39 02 26522765; Fax: +39 02 26522770; Email: debellis{at}itba.mi.cnr.it
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