title: Different approaches for noninvasive prenatal diagnosis of genetic diseases based on PNA-mediated enriched PCR.
abstract: 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.
authors.affiliation: Genomic Unit for the Diagnosis of Human Pathologies, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Galbiati
authors.forename: Gabriella
authors.initials: G
authors.lastname: Restagno
authors.forename: Barbara
authors.initials: B
authors.lastname: Foglieni
authors.forename: Sara
authors.initials: S
authors.lastname: Bonalumi
authors.forename: Maurizio
authors.initials: M
authors.lastname: Travi
authors.forename: Antonio
authors.initials: A
authors.lastname: Piga
authors.forename: Luca
authors.initials: L
authors.lastname: Sbaiz
authors.forename: Marcella
authors.initials: M
authors.lastname: Chiari
authors.forename: Francesco
authors.initials: F
authors.lastname: Damin
authors.forename: Maddalena
authors.initials: M
authors.lastname: Smid
authors.forename: Luca
authors.initials: L
authors.lastname: Valsecchi
authors.forename: Federica
authors.initials: F
authors.lastname: Pasi
authors.forename: Augusto
authors.initials: A
authors.lastname: Ferrari
authors.forename: Maurizio
authors.initials: M
authors.lastname: Ferrari
authors.forename: Laura
authors.initials: L
authors.lastname: Cremonesi
mesh_terms: D004247:DNA / Q000097:blood; D004252:DNA Mutational Analysis; D030342:Genetic Diseases, Inborn / Q000175:diagnosis*; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D020135:Peptide Nucleic Acids / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011296:Prenatal Diagnosis / Q000379:methods*; D017422:Sequence Analysis, DNA
chemical_list: D020135:Peptide Nucleic Acids; D004247:DNA