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

被引:9
作者
Bordoni, R
Consolandi, C
Castiglioni, B
Busti, E
Bernardi, LR
Battaglia, C
De Bellis, G
机构
[1] CNR, Ist Tecnol Biomed, I-20090 Segrate, Italy
[2] Univ Milan, Dipartimento Sci & Tecnol Biomed, I-20090 Segrate, Italy
[3] LITA, CISI, I-20090 Segrate, Italy
关键词
D O I
10.1093/nar/30.8.e34
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:E34 / 34
页数:7
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