title: SNP genotyping using microsphere-linked PNA and flow cytometric detection.
abstract: BACKGROUND
  Single nucleotide polymorphisms (SNPs) represent the most frequent form of genetic variations. Some of the most sensitive methods for SNP genotyping employ synthetic oligonucleotides, such as the peptide nucleic acid (PNA). We introduce a new method combining allele-specific hybridization, PNA technology, and flow cytometric detection. We tested the design by genotyping a Danish basal cell carcinoma cohort of 80 individuals for an A/C SNP in exon 6 of the XPD gene.
  
  
  METHODS
  Genomic DNA was amplified by a two-step polymerase chain reaction (PCR) in the presence of fluorescein-dyed primers and fluorescein-12-dUTP. The allele-specific PNA molecules were covalently coupled to carboxylated microspheres with and without rhodamine. Allele-specific hybridization between PCR products and immobilized PNA was carried out at 60 degrees C followed by flow cytometric detection.
  
  
  RESULTS
  We present a fully functional two-bead genotyping system based on PNA capture and flow cytometric detection used for the correct and fast regenotyping of a Danish basal cell carcinoma cohort.
  
  
  CONCLUSIONS
  This new assay presents a simple, rapid, and robust method for SNP genotyping for laboratories equipped with a standard flow cytometer. Moreover, this system offers potential for multiplexing and will be operational for middle-scale genotyping.
authors.affiliation: Institute for Human Genetics, University of Aarhus, Aarhus, Denmark.
authors.forename: Eszter
authors.initials: E
authors.lastname: Rockenbauer
authors.forename: Kenneth
authors.initials: K
authors.lastname: Petersen
authors.forename: Ulla
authors.initials: U
authors.lastname: Vogel
authors.forename: Lars
authors.initials: L
authors.lastname: Bolund
authors.forename: Steen
authors.initials: S
authors.lastname: Kølvraa
authors.forename: Kirsten Vang
authors.initials: KV
authors.lastname: Nielsen
authors.forename: Bjørn A
authors.initials: BA
authors.lastname: Nexø
mesh_terms: D002280:Basal Cell Carcinoma / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004265:DNA Helicases / Q000235:genetics; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods*; D019793:Fluorescein / Q000737:chemistry; D005838:Genotype; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D008863:Microspheres; D009693:Nucleic Acid Hybridization / Q000379:methods; D020135:Peptide Nucleic Acids / Q000737:chemistry* / Q000235:genetics; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D013696:Temperature; D014157:Transcription Factors / Q000235:genetics; D051759:Xeroderma Pigmentosum Group D Protein
chemical_list: D015342:DNA Probes; D004268:DNA-Binding Proteins; D020135:Peptide Nucleic Acids; D014157:Transcription Factors; D004247:DNA; D004265:DNA Helicases; D051759:Xeroderma Pigmentosum Group D Protein; C497292:ERCC2 protein, human; D019793:Fluorescein