title: Efficiency, error and yield in light-directed maskless synthesis of DNA microarrays.
abstract: BACKGROUND
  Light-directed in situ synthesis of DNA microarrays using computer-controlled projection from a digital micromirror device--maskless array synthesis (MAS)--has proved to be successful at both commercial and laboratory scales. The chemical synthetic cycle in MAS is quite similar to that of conventional solid-phase synthesis of oligonucleotides, but the complexity of microarrays and unique synthesis kinetics on the glass substrate require a careful tuning of parameters and unique modifications to the synthesis cycle to obtain optimal deprotection and phosphoramidite coupling. In addition, unintended deprotection due to scattering and diffraction introduce insertion errors that contribute significantly to the overall error rate.
  
  
  RESULTS
  Stepwise phosphoramidite coupling yields have been greatly improved and are now comparable to those obtained in solid phase synthesis of oligonucleotides. Extended chemical exposure in the synthesis of complex, long oligonucleotide arrays result in lower--but still high--final average yields which approach 99%. The new synthesis chemistry includes elimination of the standard oxidation until the final step, and improved coupling and light deprotection. Coupling Insertions due to stray light are the limiting factor in sequence quality for oligonucleotide synthesis for gene assembly. Diffraction and local flare are by far the largest contributors to loss of optical contrast.
  
  
  CONCLUSIONS
  Maskless array synthesis is an efficient and versatile method for synthesizing high density arrays of long oligonucleotides for hybridization- and other molecular binding-based experiments. For applications requiring high sequence purity, such as gene assembly, diffraction and flare remain significant obstacles, but can be significantly reduced with straightforward experimental strategies.
authors.affiliation: Institute of Inorganic Chemistry, University of Vienna, Währinger Strasse 42, A-1090 Vienna, Austria.
authors.forename: Christy
authors.initials: C
authors.lastname: Agbavwe
authors.forename: Changhan
authors.initials: C
authors.lastname: Kim
authors.forename: DongGee
authors.initials: D
authors.lastname: Hong
authors.forename: Kurt
authors.initials: K
authors.lastname: Heinrich
authors.forename: Tao
authors.initials: T
authors.lastname: Wang
authors.forename: Mark M
authors.initials: MM
authors.lastname: Somoza
mesh_terms: D001483:Base Sequence; D005898:Glass / Q000737:chemistry; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D009841:Oligonucleotides / Q000138:chemical synthesis*; D010777:Photochemistry / Q000379:methods; D060327:Solid-Phase Synthesis Techniques / Q000379:methods*
chemical_list: D009841:Oligonucleotides