Annals of the New York Academy of Sciences · 2006 · 13 citations · 9 references
MedicineGeneticsMolecular GeneticsGenomicsMedical GeneticsMolecular DiagnosticsMutation AnalysisMutation ScreeningNoninvasive Prenatal TestingPolymerase Chain ReactionPrenatal Genetic DiagnosisPrenatal Molecular TestingAllele-specific DetectionAmplification-based Mutation DetectionGenetic Mutation DetectionMutant DetectionPolymerase Chain Reaction (Pcr)-based DiagnosisPolymerase Chain Reaction-based Prenatal DiagnosisPolymerase Chain Reaction-based TestingSingle-nucleotide Variant Detection
The aim of this work was to develop advanced and accessible protocols for noninvasive prenatal diagnosis of genetic diseases. We are evaluating different technologies for mutation detection, based on fluorescent probe hybridization of the amplified product and pyrosequencing, a technique that relies on the incorporation of nucleotides in a primer-directed polymerase extension reaction. In a previous investigation, we have already proven that these approaches are sufficiently sensitive to detect a few copies of a minority-mutated allele in the presence of an excess of wild-type DNA, In this work, in order to further enhance the sensitivity, we have employed a mutant enrichment amplification strategy based on the use of peptide nucleic acids (PNAs). These DNA analogues bind wild-type DNA, thus interfering with its amplification while still allowing the mutant DNA to become detectable. We have synthesized different PNAs, which are highly effective in clamping wild-type DNA in the beta-globin gene region, where four beta-thalassemia mutations are located (IVSI.110, CD39, IVSI.1, IVSI.6) plus HbS. The fluorescence microchip readout allows us to monitor the extent of wild-type allele inhibition, thus facilitating the assessment of the optimal PNA concentration.
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Single base pair mutation analysis by PNA directed PCR clamping
Henrik Ørum, Peter E. Nielsen, Rolf H. Berg et al. · Nucleic Acids Research · 1993 · 337 citations · Full text