title: Detection of the R553X DNA single point mutation related to cystic fibrosis by a "chiral box" D-lysine-peptide nucleic acid probe by capillary electrophoresis.
abstract: In order to recognize the presence of the R553X point mutation of the cystic fibrosis (CF) gene in the human genome, a peptide nucleic acid (PNA) complementary to the mutated gene tract and bearing three adjacent chiral monomers based on D-lysine (chiral box) was synthesized and used as a probe in CE. Binding specificity was preliminarily studied with complementary and mismatched oligonucleotides by UV spectroscopy, electrospray MS, and electrophoresis, indicating a very high sequence selectivity. The chiral PNA probe was then hybridized to cyanine-5-labeled DNA samples (186 bp), obtained by PCR amplification, respectively, from: (a) normal homozygous subjects (wtDNA), (b) CF-affected homozygous subjects (mutDNA), (c) heterozygous subjects (healthy carriers) and denatured at low ionic strength. The PNA-DNA mixture was directly analyzed by CE with LIF detection: a new signal corresponding to the PNA-mutDNA duplex was observed, in the case of CF-affected homozygous subjects, whereas for the sample containing the mismatched sequence (normal homozygous wtDNA) only the signal corresponding to ssDNA (ss, single strand) was detected. In the case of heterozygous DNA, both PNA-mutDNA duplex and ssDNA were detected. With this simple assay, it was possible to discriminate in an easy way among the three cases (mutated homozygous, normal homozygous, and heterozygous subjects) with a total specificity, thus allowing a decisive advance for the diagnosis of CF.
authors.affiliation: Dipartimento di Chimica Organica e Industriale, Università di Parma, Parco Area delle Scienze, Italy.
authors.forename: Tullia
authors.initials: T
authors.lastname: Tedeschi
authors.forename: Marcella
authors.initials: M
authors.lastname: Chiari
authors.forename: Gianni
authors.initials: G
authors.lastname: Galaverna
authors.forename: Stefano
authors.initials: S
authors.lastname: Sforza
authors.forename: Marina
authors.initials: M
authors.lastname: Cretich
authors.forename: Roberto
authors.initials: R
authors.lastname: Corradini
authors.forename: Rosangela
authors.initials: R
authors.lastname: Marchelli
mesh_terms: D003550:Cystic Fibrosis / Q000235:genetics*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D015342:DNA Probes; D019075:Electrophoresis, Capillary; D006801:Humans; D008239:Lysine / Q000737:chemistry; D009693:Nucleic Acid Hybridization / Q000379:methods; D020135:Peptide Nucleic Acids / Q000737:chemistry*; D017354:Point Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D013237:Stereoisomerism
chemical_list: C505032:CFTR protein, human; D015342:DNA Probes; D020135:Peptide Nucleic Acids; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D008239:Lysine