DNA design and manipulation applied to the proximity ligation assay

Abstract: The proximity ligation assay is a sensitive method permitting the detection of proteins in the single event regime, by increasing the specificity of the detection using two antibodies. The detection itself is then based on DNA amplification of a circular ssDNA construct, by a DNA polymerase. This ssDNA construct is assembled on the specific oligonucleotides, previously conjugated to the antibodies. The aim of this dissertation is to allow the detection of a large number of parallel events, by investigating a method to create new DNA sequences for the conjugated oligonucleotides.
To achieve this, we developed an in silico algorithm to identify the best DNA sequence, which will allow multiple parallel PLA events to assemble without cross-interferences. The algorithm generates a library of possible candidates, and discards the ones hybridizing strongly to any sequence other than their reverse complement. The final result is a library of oligonucleotides, which can be used in the same reaction to detect different specific targets.
To validate this process, we used a mLSI platform to assess how much PLA signal is created in presence of 4 different oligonucleotides. We tested a library of 10 PLA sets, assembling every combination of the 4 fragments. This validation confirmed that the in silico algorithm optimized the correct parameters, and that the library can be used for PLA. A similar validation experiment was also designed to test a solid-phase PLA on the same mLSI platform, assembling separately every combination of the 4 oligonucleotides, and measuring the signal from the PLA method.
Having validated the development of the different sets of oligonucleotides for the PLA, we made an experiment in situ on human cells, trying to overcome the limitation of the 3 parallel fluorescent detection probes, by erasing the DNA signal between different rounds of detection. At this step, we discovered that the polymerase exonuclease activity causes degradation of any ssDNA fragment present in the cells, including unreacted oligonucleotides bound to the antibodies.
To overcome this limitation, a new PLA setup was designed to use next generation sequencing as readout. A new process was then designed, where the product of the DNA amplification is digested using a restriction enzyme. The library was then recovered from the chip, ready for sequencing. New oligonucleotides were designed to include a single restriction site on the circular template, as well as identifiers included in the template. This design enabled an increase in the identification depth from identical PLA events to single PLA events.
The complete process highlights the strength of in silico design of DNA sequence based on minimal free energy, in a relatively new field of entirely artificial DNA assembly. This is different than hybridization of ssDNA to existing DNA for detection, like any PCR, padlock probes, sequencing or many RNA techniques

Angaben zum Objekt

• Standort: Deutsche Nationalbibliothek Frankfurt am Main
• Umfang: Online-Ressource
• Sprache: Englisch
• Anmerkungen: Universität Freiburg, Dissertation, 2017

Klassifikation und Themen

• Schlagwort: Design ; Bar coding ; DNA Barcoding ; DNA-Sensor ; Immunoblot ; Bioinformatik ; Bioinformatikstudium ; In silico-Methode ; Molekulare Bioinformatik ; Microarray ; Mikrofluidik ; High throughput screening ; Massive parallele Sequenzierung

Beteiligte, Orts- und Zeitangaben

• Ereignis: Veröffentlichung
• (wo): Freiburg
• (wer): Universität
• (wann): 2020
• Urheber: Gobet, Nicolas
• Beteiligte Personen und Organisationen: Meier, Matthias ; Albert-Ludwigs-Universität Freiburg. Fakultät für Angewandte Wissenschaften

Weitere Informationen

• DOI: 10.6094/UNIFR/167281
• URN: urn:nbn:de:bsz:25-freidok-1672815
• Rechteinformation: Der Zugriff auf das Objekt ist unbeschränkt möglich.
• Letzte Aktualisierung: 25.03.2025, 13:48 MEZ