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Methods for the analysis of bio-molecules using rolling circle DNA synthesis

Jakob Schwalbe Lohmann  


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Accepted by: Faculty of Health Sciences University of Aarhus
Defended on: January 18, 2008
Official opponents: Professor, dr.med. Thomas Lede , Professor, dr.med. Niels Tommerup , dr.med. Eigil Kjeldsen
Tutors: Associate Professor Jørn Koch , Professor Stephen Hamilton-Dutoit. Ph.d.

Published in the PhD Database: January 24, 2008


English abstract
ABSTRACT
This PhD project was mainly carried out in the Laboratory of Molecular Pathology, Institute of Pathology, Aarhus University Hospital and funded by the EU-framework 6 project Moltools (www.Moltools.org), which was set to develop new molecular tools. In this particular thesis we have aimed at I) Develop new methods for the detection of DNA in situ. II) Improve the quality of the DNA probes used and III) Develop new methods for the detection of the activity of DNA modifying enzymes. All of these projects used rolling circle replication (RCR) of short circular DNA oligonucleotides as the basic tool. In this technique a DNA circle is replicated resulting in a long single stranded DNA sequence consisting of tandem repeats of the complementary sequence of the original circle. This product can e.g. be detected by in situ hybridization of short fluorescently labeled oligonucleotides.

Under part I, we have been able to detect short repetitive genomic DNA sequences in situ on condensed metaphase chromosomes as well as the apolipoprotein(a) gene using padlock probes and target primed RCR . Similar results had previously only been obtained on mitochondrial DNA with the setup we have used. This work was published in BMC Molecular Biology.

The quality of long chemically synthesized oligonucleotides is normally not impressive. Under part II, we have developed a method for the enzymatic production of DNA oligonucleotides using so-called ¿suicide cassettes¿, which is a hairpin structure containing binding and cleavage sites for both a nicking enzyme and a restriction enzyme. Hairpin-containing oligonucleotides were circularized and amplified by RCR and nicked with a nicking enzyme. This results in that the tandem repeat is turned into monomers, which can be re-circularized and again amplified by RCR. These steps can be performed numerous times, allowing for a massive amplification. In the final step the hairpin can be removed by cleavage with the restriction enzyme Mly I, which cleaves blunt end outside its recognition sequence, allowing for the release of the DNA sequence contained within the suicide cassette. This work was published in BMC Biotechnology.

Rolling circle replication has previously been used as an amplification step for the detection of DNA, RNA and protein. Using the molecular design derived under part II, we introduce under part III a new method for the detection of protein activity of DNA modifying enzymes. The method is based on enzymatic circularization of DNA oligonucleotides, which can be detected by RCR. We have detected activity of topoisomerase I and Fen1 using purified enzymes. This method may be a useful tool in determining the activity of DNA modifying enzymes or enzymatic pathways. This work has been submitted, but not yet published.



Danish abstract
Det eksperimentelle arbejde, der danner grundlag for denne ph.d.-afhandling, er hovedsagligt blevet udført i laboratoriet for Molekylær Patologi, Patologisk Institut Aarhus Universitetshospital. Projektet bygger på en bevilling under EU¿s 6. rammeprogram Moltools (www.moltools.org). Formålet med projektet var at udvikle nye metoder til detektion af enkelt-molekyler i celle- og vævs-præparationer. Dette skulle gøres ved at bruge en speciel amplifikations-teknik, der benytter små enkeltstrengede DNA-cirkler (ca. 100 nukleotider). Når sådanne cirkler er blevet dannet, kan de amplificeres meget kraftigt ved hjælp af en polymerase, og efterfølgende kan et amplifikations-produkt detekteres med fluorescence-mikroskopi. Resultaterne er blevet præsenteret i tre separate videnskabelige rapporter.

I den første del af projektet viste vi, at man kan detektere to repeterede genomiske sekvenser på kromosom-spredninger ved hjælp af såkaldte padlock-prober og rullende cirkler. De to sekvenser var henholdsvis satellit I sekvensen (DYZ1), der er placeret på den lange arm af Y-kromosomet, og det repeterede kringle IV domæne i apolipoprotein(a)-genet, der er placeret på den lange arm af kromosom 6. Denne teknik er ikke tidligere blevet eftervist på kromosom-spredninger. Dette studie er blevet publiceret i tidsskriftet BMC Molecular Biology.

I den anden del af projektet prøvede vi at forbedre de prober der blev anvendt i det første studie, da kemisk syntetiserede DNA prober sjældent har den samme kvalitet som enzymatisk syntetiserede prober. Vi har derfor udviklet en metode, der benytter en kombination af rullende cirkler samt restriktions- og nicking-enzymer til at producere padlock prober. Dette studie er blevet publiceret i tidsskriftet BMC Biotechnology.

Rullende cirkler er tidligere blevet brugt til at detektere tilstedeværelsen af DNA, RNA og proteiner, men ikke til detektion af protein-aktivitet. I den tredje del af projektet har vi udviklet en metode til netop detektion af enzymaktivitet af DNA-modificerende enzymer. Som test-proteiner har vi kigget på nukleasen Fen1 samt topoisomerase I. Metoden kan have store anvendelsesmæssige perspektiver, da det i mange henseender vil være hensigtsmæssigt at kigge på den enzymatiske aktivitet frem for alene at kigge på tilstedeværelsen af et protein. Dette gør sig gældende da de fleste proteiner bliver reguleret efter syntesen, og derved kan have forskellige enzymatiske aktiviteter afhængig af graden af regulering. Reguleringen kan ske både gennem interaktion med andre proteiner, samt post-translationelle modifikationer. Dette arbejde er indsendt men endnu ikke publiceret.