Abstract
Amine-modified oligodeoxynucleotides (AMO) are commonly used probe oligodeoxynucleotides for DNA microarray preparation. Two methods are currently used for AMO preparation—use of amine phosphoramidites protected by acid-labile monomethoxytrityl (MMT) groups or alkali-labile trifluoroacetyl (TFA) groups. Because conventional AMO preparation procedures have defects, for example stringent acidic conditions are required for deprotection of MMT and hydrophobic purification cannot be used for TFA-protected amino groups, conventional preparation of AMO is unlikely to result in the expected outcome. In this paper a method of AMO synthesis using modified H-phosphonate chemistry is suggested. An aliphatic diamine is coupled with a phosphonate group forming a phosphoramidate linkage to the last internucleotide phosphate of oligodeoxynucleotides. In this method dimethoxytrityl (DMT) purification steps are used and stringent acid deprotection is not required to obtain the AMO. Although the method could lead to formation of AMO diastereomers, melting-temperature and CD analysis showed for two AMO that DNA duplex formation was the same as when normal oligodeoxynucleotides were used. Also, when these AMO were used as probes for DNA microarrays the immobilization efficiency was similar to that for AMO probes prepared by conventional means using an amino-modifier unit. The hybridization performance of these AMO was better than for those prepared conventionally. The procedures suggested would be useful for preparation of efficient AMO for fabrication of DNA microarrays and DNA-based nanoparticle systems.
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Acknowledgement
This work is partially supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan, to K.M. (No. 18350083) and by Grants-in-Aid for regional science and technology promotion the “Kyoto Nanotechnology Cluster” project from MEXT, Japan. This work was also supported by CREST of the Japan Science and Technology Agency. We also thank Yasuko Yoshida and Kazunari Yamada (NGK Insulators Ltd, Geneshot Project, Japan) for providing an ink-jet spotter and image scanners for acquiring quantitative fluorescence data. This work was also supported by the Korean Research Foundation (No. M01-2005-000-10436-0).
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Nagendra Kumar Kamisetty and Seung Pil Pack have equally contributed to this work.
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Kamisetty, N.K., Pack, S.P., Nonogawa, M. et al. Efficient preparation of amine-modified oligodeoxynucleotide using modified H-phosphonate chemistry for DNA microarray fabrication. Anal Bioanal Chem 387, 2027–2035 (2007). https://doi.org/10.1007/s00216-006-1097-7
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DOI: https://doi.org/10.1007/s00216-006-1097-7