title: Express photolithographic DNA microarray synthesis with optimized chemistry and high-efficiency photolabile groups.
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.
authors.affiliation: Institute of Inorganic Chemistry, Faculty of Chemistry, University of Vienna, Vienna, Austria. matej.sack@univie.ac.at.
authors.forename: Matej
authors.initials: M
authors.lastname: Sack
authors.affiliation: Institute of Inorganic Chemistry, Faculty of Chemistry, University of Vienna, Vienna, Austria. kathrin.hoelz@univie.ac.at.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Hölz
authors.affiliation: Department of Nutritional and Physiological Chemistry, Faculty of Chemistry, University of Vienna, Vienna, Austria. ann-katrin.holik@univie.ac.at.
authors.forename: Ann-Katrin
authors.initials: AK
authors.lastname: Holik
authors.affiliation: Institute of Inorganic Chemistry, Faculty of Chemistry, University of Vienna, Vienna, Austria. nicole.kretschy@univie.ac.at.
authors.forename: Nicole
authors.initials: N
authors.lastname: Kretschy
authors.affiliation: Department of Nutritional and Physiological Chemistry, Faculty of Chemistry, University of Vienna, Vienna, Austria. veronika.somoza@univie.ac.at.|Christian Doppler Laboratory for Bioactive Aroma Compounds, University of Vienna, Vienna, Austria. veronika.somoza@univie.ac.at.
authors.forename: Veronika
authors.initials: V
authors.lastname: Somoza
authors.affiliation: Roche Diagnostics, Penzberg, Germany. klaus-peter.stengele@roche.com.
authors.forename: Klaus-Peter
authors.initials: KP
authors.lastname: Stengele
authors.affiliation: Institute of Inorganic Chemistry, Faculty of Chemistry, University of Vienna, Vienna, Austria. mark.somoza@univie.ac.at.
authors.forename: Mark M
authors.initials: MM
authors.lastname: Somoza
mesh_terms: D018938:Caco-2 Cells; D045744:Cell Line, Tumor; D004247:DNA / Q000737:chemistry*; D006801:Humans; D008027:Light; D009693:Nucleic Acid Hybridization / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D009841:Oligonucleotides / Q000737:chemistry*; D009943:Organophosphorus Compounds / Q000737:chemistry; D010777:Photochemistry / Q000379:methods; D060327:Solid-Phase Synthesis Techniques / Q000379:methods
chemical_list: D009841:Oligonucleotides; D009943:Organophosphorus Compounds; C434331:phosphoramidite; D004247:DNA