Characterization and sequence verification of thiolated deoxyoligonucleotides used for microarray construction.

During synthesis of thiolated deoxyoligonucleotides, side products can be formed. When used in the fabrication of microchips, the oligonucleotides have to be of high purity. We demonstrated the presence of impurities, which were not failure sequences from the synthesis. These products were identified and characterized using high-performance liquid chromatography (HPLC) and electrospray MS(/MS). The presence of the free thiol group was assessed by acrylamide derivatization. After reaction with acrylamide the correct compounds showed a 71 u mass shift and the major fragment ions could be assigned as 5' a-base and 3' w ions, similar as for unmodified DNA. The side products were unaffected by acrylamide, suggesting that the thiol group was modified. The oligonucleotide containing a species with a mass of 32 u higher was identified as 5' sulfinic acid containing molecule and was found as 45% of the total amount of a DNA 25 mer. Other components appeared to be dithio-linked oxidation products, present about 1 to 5% in a 10 mer and 15 mer deoxyoligonucleotide. The analyses were useful for optimizing the synthesis protocols.