Parallel synthesis of oligonucleotide microarrays using photogenerated acids and digital photolithography, and investigation of DNA synthesis fidelity on flat solid surfaces

DNA microarrays have emerged as a powerful tool in numerous genomic applications such as gene expression and genotyping applied towards disease understanding and drug development. However, the fabrication of such miniaturized devices faces several challenges, and the major among those are cost and quality. As a result, the synthesis fidelity of DNA on flat solid surface was scrutinized. Our investigation revealed an improved analytical method for the characterization of in situ synthesis fidelity using ammonolysis cleavage, 32P labeling and gel electrophoresis, and its application to the determination of individual stepwise yields of the in situ DNA synthesis cycle. In addition, “hindered sites” on the surface were identified as the cause of poor synthesis yield, and an alternative OH linker was introduced as a new attachment point for in situ DNA synthesis on flat solid surface.; A novel oligonucleotide DNA synthesis was also developed using photogenerated acids in solution and digital photolithography to affect otherwise conventional, parallel in situ DNA synthesis. First, it was demonstrated that PGAs were compatible with the detritylation of DNA attached on CPG, as well as with the subsequent phosphoramidite chemistry. Second, the synthesis of patterned microarray substrates was optimized to physically isolated droplets of PGA solution during the light events. Third, a multidimensional search for optimized PGA solution conditions compatible with the light requirements of the DNA synthesis platform was conducted. The development of a combinatorial screening method using a fluorescent dye as reporter of detritylation efficiency expedited the process. Fourth, the detritylation efficiency of the selected PGA was characterized using a similar combinatorial approach. This study revealed that acid formation was fast, completed within a few seconds, and that response to light irradiation was non linear. Fifth, the synthesis of oDNA microarrays was performed and characterized by hybridization to fluorescently labeled complementary DNA targets. Single base pair mismatch discrimination assays were performed and demonstrated that not only were the correct sequences synthesized, but also that any mismatches could be discriminated.