Improved oligonucleotides for microarrays

Under the conditions tested, the authors report that the best yield of inverted oligonucleotides represented about 25% of the maximum amount of material that could have been synthesized. Analysis of inversion products following in situ synthesis of mixed-length oligonucleotides, which mimic truncated synthesis products, indicated that the inversion process resulted in effective in situ oligonucleotide purification by eliminating truncated products from the solid support. The inverted products were further tested for function in minisequencing and pyrosequencing assays. Both assays indicated that DNA polymerase could add nucleotides to the 3' -OH end of an oligonucleotide resulting from inversion.There are extensive descriptions of the innovative chemical derivatization of the solid support and of the in situ synthesis protocols which enable the inversion of oligonucleotides. Because ''synthesis on planar solid supports results in a limited quantity of product'', the authors used 50-70 μm diameter polystyrene beads to obtain sufficient product to monitor the individual steps of the inversion procedure. The 5' functionality that allowed inversion was an added o-chlorophenyl phosphodiester moiety, which in the presence of a condensing agent forms a phosphodiester linkage between the 5' end of the oligonucleotide and the solid support. Analysis by capillary electrophoresis of reaction products cleaved from the solid support indicated that two major products were obtained after inversion - a mix of 5' inverted and non-inverted 19 base oligonucleotides (19mers). The 5' inverted 19mers occupied a physically distinct peak on the chromatogram as a result of the attachment of several triethyleneglycol moieties, added to facilitate cleavage of the oligonucleotides from the solid support and to distinguish inverted oligonucleotides from truncated products.According to the authors, this method is suitable for the production of oligonucleotide arrays and is compatible with all ex