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Gobet, N.* ; Ketterer, S.* ; Meier, M.

Design and validation of DNA libraries for multiplexing proximity ligation assays.

PLoS ONE 9:e112629 (2014)

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Open Access Gold as soon as Publ. Version/Full Text is submitted to ZB.

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Here, we present an in silico, analytical procedure for designing and testing orthogonal DNA templates for multiplexing of the proximity ligation assay (PLA). PLA is a technology for the detection of protein interactions, post-translational modifications, and protein concentrations. To enable multiplexing of the PLA, the target information of antibodies was encoded within the DNA template of a PLA, where each template comprised four single-stranded DNA molecules. Our DNA design procedure followed the principles of minimizing the free energy of DNA cross-hybridization. To validate the functionality, orthogonality, and efficiency of the constructed template libraries, we developed a high-throughput solid-phase rolling-circle amplification assay and solid-phase PLA on a microfluidic platform. Upon integration on a microfluidic chip, 640 miniaturized pull-down assays for oligonucleotides or antibodies could be performed in parallel together with steps of DNA ligation, isothermal amplification, and detection under controlled microenvironments. From a large computed PLA template library, we randomly selected 10 template sets and tested all DNA combinations for cross-reactivity in the presence and absence of antibodies. By using the microfluidic chip application, we determined rapidly the false-positive rate of the design procedure, which was less than 1%. The combined theoretical and experimental procedure is applicable for high-throughput PLA studies on a microfluidic chip.

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Publication type Article: Journal article

Document type Scientific Article

Corresponding Author

ISSN (print) / ISBN 1932-6203

Journal PLoS ONE

Quellenangaben Volume: 9, Issue: 11, Article Number: e112629

Publisher Public Library of Science (PLoS)

Publishing Place Lawrence, Kan.

Non-patent literature Publications

Reviewing status Peer reviewed

Institute(s) Helmholtz Pioneer Campus (HPC)