Proximity Ligation : Transforming protein analysis into nucleic acid detection through proximity-dependent ligation of DNA sequence tagged protein-binders
Sammanfattning: A novel technology for protein detection, proximity ligation, has been developed along with improved methods for in situ synthesis of DNA microarrays. Proximity ligation enables a specific and quantitative transformation of proteins present in a sample into nucleic acid sequences. As pairs of so-called proximity probes bind the individual target protein molecules at distinct sites, these reagents are brought in close proximity. The probes consist of a protein specific binding part coupled to an oligonucleotide with either a free 3’- or 5’-end capable of hybridizing to a common connector oligonucleotide. When the probes are in proximity, promoted by target binding, then the DNA strands can be joined by enzymatic ligation. The nucleic acid sequence that is formed can then be amplified and quantitatively detected in a real-time monitored polymerase chain reaction. This convenient assay is simple to perform and allows highly sensitive protein detection. Parallel analysis of multiple proteins by DNA microarray technology is anticipated for proximity ligation and enabled by the information carrying ability of nucleic acids to define the individual proteins. Assays detecting cytokines using SELEX aptamers or antibodies, monoclonal and polyclonal, are presented in the thesis.Microarrays synthesized in situ using photolithographic methods generate impure products due to damaged molecules and interrupted synthesis. Through a molecular inversion mechanism presented here, these impurities may be removed. At the end of synthesis, full-length oligonucleotides receive a functional group that can then be made to react with the solid support forming an arched structure. The 3’-ends of the oligonucleotides are then cleaved, removing the impurities from the support and allowing the liberated 3’-hydroxyl to prime polymerase extension reactions from the inverted oligonucleotides. The effect of having pure oligonucleotides probes compared to ones contaminated with shorter variants was investigated in allele specific hybridization reactions. Pure probes were shown to have greater ability to discriminate between matched and singly mismatched targets at optimal hybridization temperatures.
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