Characterization of oligodeoxyribonucleotide synthesis on glass plates

Achieving high fidelity chemical synthesis on glass plates has become incre asingly important, since glass plates are substrates widely used for miniat urized chemical and biochemical reactions and analyses. DNA chips can be di rectly prepared by synthesizing oligonucleotides on glass plates, but the c haracterization of these micro-syntheses has been limited by the sub-picomo lar amount of material available. Most DNA chip syntheses have been assayed using in situ coupling of fluorescent molecules to the 5'-OH of the synthe sized oligonucleotides. We herein report a systematic investigation of olig onucleotide synthesis on glass plates with the reactions carried out in an automated DNA synthesizer using standard phosphoramidite chemistry. The ana lyses were performed using P-32 gel electrophoresis of the oligonucleotides cleaved from glass plates to provide product distribution profiles accordi ng to chain length of oligonucleotides. 5'-Methoxythymidine was used as the chain terminator, which permits assay of coupling reaction yields as a fun ction of chain length growth. The results of this work reveal that a major cause of lower fidelity synthesis on glass plates is particularly inefficie nt reactions of the various reagents with functional groups close to glass plate surfaces. These problems cannot be detected by previous in situ fluor escence assays. The identification of this origin of low fidelity synthesis on glass plates should help to achieve improved synthesis for high quality oligonucleotide microarrays.