Evaluating the quality of oligonucleotides that are immobilized on glass supports for biosensor development

Three different analytical techniques were compared to assess methodologies that could evaluate the success of oligonucleotide assembly on glass substrates functionalized with hexaethylene glycol (HEG) linker molecules, Post-synthesis cleavage of the linker-oligonucleotide conjugates from the solid support was done to prepare samples for analysis, Samples were investigated by electrospray ionization mass spectrometry (ESI-MS), high performance ion-exchange liquid chromatography (HPIEC) and polyacrylamide gel electrophoresis after radiolabeling (P-32-PAGE), The data from ESI-MS served to identify the various species detected by HPIEC and 32P-PAGE. All three techniques were shown to be very sensitive to the presence and location (terminal or internucleotide) of HEG conjugated to the oligonucleotide sequence. This allowed differentiation and quantification of linker-oligonucleotides from non-conjugate oligonucleotides that originated from undesired synthesis directly on the glass surface. Furthermore, shorter linker-oligonucleotide conjugates that were formed by incomplete nucleobase-coupling during DNA synthesis on the linker could be identified by HPIEC and P-32-PAGE, allowing purity assessment of the assembled strands, Despite the inherent higher sensitivity of PAGE of radiolabeled samples, HPIEC was shown to be the method of choice due to high sample throughput and facile quantitative analysis of the products. (C) 1999 Elsevier Science B.V, All rights reserved.