Efficient preparation of amine-modified oligodeoxynucleotide using modified H-phosphonate chemistry for DNA microarray fabrication.

Nagendra Kumar Kamisetty, Seung Pil Pack, Mitsuru Nonogawa, Kamakshaiah Charyulu Devarayapalli, Seiya Watanabe, Tsutomu Kodaki, Keisuke Makino

文献索引：Anal. Bioanal. Chem 387(6) , 2027-35, (2007)

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摘要

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.