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Nucleic Acids Research, 2001, Vol. 29, No. 10 2171-2180
© 2001 Oxford University Press

Characterization of oligodeoxyribonucleotide synthesis on glass plates

Eric LeProust, Hua Zhang, Peilin Yu, Xiaochuan Zhou1 and Xiaolian Gao*

Department of Chemistry, University of Houston, Houston, TX 77204-5641, USA and 1Xeotron Co., 8285 El Rio, Suite 130, Houston, TX 77054, USA

Achieving high fidelity chemical synthesis on glass plates has become increasingly important, since glass plates are substrates widely used for miniaturized chemical and biochemical reactions and analyses. DNA chips can be directly prepared by synthesizing oligonucleotides on glass plates, but the characterization of these micro-syntheses has been limited by the sub-picomolar amount of material available. Most DNA chip syntheses have been assayed using in situ coupling of fluorescent molecules to the 5'-OH of the synthesized oligonucleotides. We herein report a systematic investigation of oligonucleotide synthesis on glass plates with the reactions carried out in an automated DNA synthesizer using standard phosphoramidite chemistry. The analyses were performed using 32P gel electrophoresis of the oligonucleotides cleaved from glass plates to provide product distribution profiles according to chain length of oligonucleotides. 5'-Methoxythymidine was used as the chain terminator, which permits assay of coupling reaction yields as a function of chain length growth. The results of this work reveal that a major cause of lower fidelity synthesis on glass plates is particularly inefficient reactions of the various reagents with functional groups close to glass plate surfaces. These problems cannot be detected by previous in situ fluorescence assays. The identification of this origin of low fidelity synthesis on glass plates should help to achieve improved synthesis for high quality oligonucleotide microarrays.

* To whom correspondence should be addressed. Tel: +1 713 743 2805; Fax: +1 713 743 2709; Email: xgao{at}uh.edu The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors


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