Biomedical subjects

Anette Jacob

Publications and source records attributed to Anette Jacob.

3 recordsLinked to original sources

PubMed2004

Peptide nucleic acid microarrays.

A fast and economical procedure for the production of peptide nucleic acid (PNA) microarrays is presented. PNA oligomers are synthesized in a fully automatic manner in 96-well plates using standard Fmoc chemistry. Subsequently, the oligomers are released from the support and spotted onto glass or silicone slides, which were activated by succinimidyl ester. This process allows for a concomitant purification of the oligomers directly on the chip surface. Although the terminal primary amino groups of the full-length products bind selectively to this surface, none of the byproducts of synthesis, such as truncated sequences or cleaved side chain protection groups, will bind and are therefore washed away. In this chapter, protocols are presented for the whole production process as well as sample hybridization.

Nucleic Acid Hybridization↗

PubMed2003

PNA microarrays for hybridisation of unlabelled DNA samples.

Several strategies have been developed for the production of peptide nucleic acid (PNA) microarrays by parallel probe synthesis and selective coupling of full-length molecules. Such microarrays were used for direct detection of the hybridisation of unlabelled DNA by time-of-flight secondary ion mass spectrometry. PNAs were synthesised by an automated process on filter-bottom microtitre plates. The resulting molecules were released from the solid support and attached without any purification to microarray surfaces via the terminal amino group itself or via modifications, which had been chemically introduced during synthesis. Thus, only full-length PNA oligomers were attached whereas truncated molecules, produced during synthesis because of incomplete condensation reactions, did not bind. Different surface chemistries and fitting modifications of the PNA terminus were tested. For an examination of coupling selectivity, bound PNAs were cleaved off microarray surfaces and analysed by MALDI-TOF mass spectrometry. Additionally, hybridisation experiments were performed to compare the attachment chemistries, with fully acetylated PNAs spotted as controls. Upon hybridisation of unlabelled DNA to such microarrays, binding events could be detected by visualisation of phosphates, which are an integral part of nucleic acids but missing entirely in PNA probes. Overall best results in terms of selectivity and sensitivity were obtained with thiol-modified PNAs on maleimide surfaces.

Base Sequence↗

PubMed2003

Antibody microarrays: an evaluation of production parameters.

Antibody microarrays could have an enormous impact on the functional analysis of cellular activity and regulation, especially at the level of protein expression and protein-protein interaction, and might become an invaluable tool in disease diagnostics. The array surface is bound to have a tremendous influence on the findings from such studies. Apart from the basic issue of how to attach antibodies optimally without affecting their function, it is also important that the cognate antigens, applied within a complex protein mixture, all bind to the arrayed antibodies irrespective of their enormous variety in structure. In this study, various factors in the production of antibody microarrays on glass support were analysed: the modification of the glass surface; kind and length of cross-linkers; composition and pH of the spotting buffer; blocking reagents; antibody concentration and storage procedures, in order to evaluate their effect on array performance. Altogether, data from more than 700 individual array experiments were taken into account. In addition to home-made slides, commercially available systems were also included in the analysis.

Absorption↗