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