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Content archived on 2024-04-30

New diagnostic system for specific and fast detection of multiple nucleic acid mutations by PNA hybridization on an array sensor


(1) Technology-related: Finding, designing and synthesis of a sensitive and PNA:NAspecific dye; fixation of PNA on useful solid supports; specific hybridisation of DNA with solid support-bound PNA; detection of PNA:NA hybrids by specific dye interaction; development of bread boards for detection.

(2) Application related: Development of a working model with the new dye using model DNA sequences related to selected p53 mutations; application of the working model to representative sS3 mutations from clinical samples.

At present, nucleic acid (NA) diagnostic systems are based on hybridisation between NA analytes and NA probes labelled with a reporter group. This type of hybridisation is limited regarding sensitivity, specificity, complexity, hybridisation conditions, and the need of prior probe labelling. To overcome these limitations new diagnostic principles for specific and fast detection of NA will be investigated which are obtained by specific hybridisation of PCR products of diagnostic relevant analyte sequences with solid support-bound PNA. In case of PNA the entire backbone has been replaced by a backbone composed of N-(2-aminoethyl)glycine units leaving the nucleobases intact. Although this alternative backbone chemically does not even remotely resemble the deoxyribose phosphodiester backbone of DNA, it is structurally homomorpheous to the DNA backbone. The PNA duplexes of mixed purine/pyrimidine sequences with Watson-Crick-like helix structures are more stable than the corresponding NA:NA duplexes, but the kinetic parameters are similar for the formation of PNA:NA and PNA:PNA duplexes. Since PNA is a pseudo-peptide, chemical modification and conjugation is fairly straight forward. Recently it has been shown that known NA:NA hybrid-interacting dyes interact in a different way with PNA:NA hybrids. Therefore one aim of this project is to find highly sensitive dyes which interact selectively only with PNA:NA-hybrids. By combining such a dye with the advantages of PNA hybridisation, the basis for a new and improved array-like diagnostic sensor will be developed in form of a model system. A prerequisite for this model system is the specific formation and detection of PNA:NA hybrids on a surface in array like geometry. For PNA analysis of genomic mutation patterns a working model with the new dye will be set up. As substrates will be used selected p53-related oligonucleotides and DNA model sequences obtained by PCR amplification from p53 plasmids. Additionally representative pS3 mutations from clinical samples will be analysed applying the model system to show the clinical potential of a PNA array sensor.

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Kobenhavns Universitet
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3 C,Blegdamsvej
2200 Kobenhavn N

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Participants (5)