DNA-kontrollierte Verknüpfung von Peptidnucleinsäuren

Abstract

The hybridization-controlled ligation of oligonucleotides is one of the fundamental approaches that is utilized in diagnostic assays such as the oligonucleotide ligation assay (OLA). The fidelity of this strategy is dependent on the sequence selectivity of the nucleic acid hybridization. The DNA-analogue peptide nucleic acid (PNA) exhibits a remarkable high binding affinity and specificity to complementary nucleic acids, making it an attractive tool for gene targeting applications. PNA is an artificial base-pairing system and biochemical tools such as ligases are do not recognize PNA as a substrate. Therefore, PNA-based ligations have to rely solely on chemical methods. For the PNA-based ligation assay for detection of single base mutations, we fashioned an approach in homogeneous solution, in which one nucleobase remains unpaired by forming an abasic site upon ligation thereby increasing the ligation fidelity. Of unique particular interest in diagnostic applications are assay systems allowing for a multiplexed detection of diagnostic events. Mass-spectrometrical detection offers unparalleled resolution and is therefore ideally suited for multiplex detection. In contrast to DNA, PNA exhibits an incomparable suitability for MALDI-TOF/MS analysis in terms of molecular weight resolution and accuracy. The efficiency of the abasic site ligation was successfully tested in a triplex competition experiment, investigating three potential mutations (G12V, F28L, E63H) in the ras gene segment. It can be concluded that the mass-spectrometrical analysis of the abasic site ligation serves as a highly specific means for the detection of single base mutations even in a competitive multiplex format.