Author:

Keywords:

Science & Technology, Life Sciences & Biomedicine, Biotechnology & Applied Microbiology, Nanoscience & Nanotechnology, Science & Technology - Other Topics, Microarray, Phosphoramidite chemistry, NPPOC, Thiophenyl-NPPOC, Photolabile, LIGHT-DIRECTED SYNTHESIS, DEOXYNUCLEOSIDE PHOSPHORAMIDITES, PROTECTING GROUPS, GENE-EXPRESSION, IN-SITU, OLIGONUCLEOTIDE SYNTHESIS, NUCLEOTIDE CHEMISTRY, CHEMICAL-SYNTHESIS, HUMAN-CELLS, ARRAYS, Caco-2 Cells, Cell Line, Tumor, DNA, Humans, Light, Nucleic Acid Hybridization, Oligonucleotide Array Sequence Analysis, Oligonucleotides, Organophosphorus Compounds, Photochemistry, Solid-Phase Synthesis Techniques, 10 Technology, 3001 Agricultural biotechnology, 3106 Industrial biotechnology, 3206 Medical biotechnology

Abstract:

BACKGROUND: DNA microarrays are a core element of modern genomics research and medical diagnostics, allowing the simple and simultaneous determination of the relative abundances of hundreds of thousands to millions of genomic DNA or RNA sequences in a sample. Photolithographic in situ synthesis, using light projection from a digitally-controlled array of micromirrors, has been successful at both commercial and laboratory scales. The advantages of this synthesis method are its ability to reliably produce high-quality custom microarrays with a very high spatial density of DNA features using a compact device with few moving parts. The phosphoramidite chemistry used in photolithographic synthesis is similar to that used in conventional solid-phase synthesis of oligonucleotides, but some unique differences require an independent optimization of the synthesis chemistry to achieve fast and low-cost synthesis without compromising microarray quality. RESULTS: High microarray quality could be maintained while reducing coupling time to a few seconds using DCI activator. Five coupling activators were compared, which resulted in microarray hybridization signals following the order ETT > Activator 42 > DCI ≫ BTT ≫ pyridinium chloride, but only the use of DCI led to both high signal and highly uniform feature intensities. The photodeprotection time was also reduced to a few seconds by replacing the NPPOC photolabile group with the new thiophenyl-NPPOC group. Other chemical parameters, such as oxidation and washing steps were also optimized. CONCLUSIONS: Highly optimized and microarray-specific phosphoramidite chemistry, along with the use of the very photosensitive thiophenyl-NPPOC protecting group allow for the synthesis of high-complexity DNA arrays using coupling times of 15 s and deprotection times of 9 s. The resulting overall cycle time (coupling to coupling) of about 50 s, results in a three-fold reduction in synthesis time.