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.

Figure 1

(A) The hairpin-structure oligonucleotide is composed of four elements: a loop comprising six adenosines with five, three or one phosphorothioate moieties, a double-strand stem 8 (5′-CCTGGCGC-3′) or 5 (5′-CCTGG-3′) bp long, three adenosines and the probe sequence (5′-TTGTACACACCGCCCGTCACGT-3′). (B) The linear structure oligonucleotide composed of a 5′ arm with different number and position of phosphorothioate modifications (A*) and the same probe sequence (5′-TTGTACACACCGCCCGTCACGT-3′).

Figure 2

Enzymatic reaction scheme of solid-phase ligation reaction. (A) Molecular interactions; (B) detection ligation product by microarray.

Figure 3

Solid-phase ligation reaction onto the APMDES (A) and GOPS (B) surfaces. Spots image (on the left) and average and standard deviation graph (on the right) of 40 replicates.

Figure 4

Solid-phase ligation reactions onto the GOPS surface with the biological sample such as 16S rRNA gene PCR product. (A) Spots image; (B) average and standard deviation graph of 40 replicates.

Figure 5

Comparison between oligonucleotide structures with a variable number of phosphorothioate moieties. 1A*,3A*,5A* and 10A* represent oligonucleotides with one, three, five and 10 phosphorothioate moieties, respectively.