Efficiency, error and yield in light-directed maskless synthesis of DNA microarrays
Autor: | Dong Gee Hong, Kurt W. Heinrich, Mark M. Somoza, Tao Wang, Changhan Kim, Christy Agbavwe |
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Jazyk: | angličtina |
Rok vydání: | 2011 |
Předmět: |
Materials science
lcsh:Medical technology Photochemistry lcsh:Biotechnology Oligonucleotides Biomedical Engineering Medicine (miscellaneous) Pharmaceutical Science Bioengineering Oligonucleotide synthesis Microarray 010402 general chemistry 01 natural sciences Chemical synthesis Applied Microbiology and Biotechnology Solid-phase synthesis lcsh:TP248.13-248.65 Solid-Phase Synthesis Techniques Oligonucleotide Array Sequence Analysis gene synthesis Phosphoramidite Base Sequence DNA synthesis 010405 organic chemistry Oligonucleotide Research NPPOC Nucleic Acid Hybridization phosphoramidite chemistry Combinatorial chemistry 0104 chemical sciences Kinetics lcsh:R855-855.5 Yield (chemistry) Molecular Medicine Glass |
Zdroj: | Journal of Nanobiotechnology, Vol 9, Iss 1, p 57 (2011) Journal of Nanobiotechnology |
ISSN: | 1477-3155 |
Popis: | 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. |
Databáze: | OpenAIRE |
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