SNP genotyping using microsphere-linked PNA and flow cytometric detection.
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.
Rockenbauer
Eszter
E
Institute for Human Genetics, University of Aarhus, Aarhus, Denmark.
Petersen
Kenneth
K
Vogel
Ulla
U
Bolund
Lars
L
Kølvraa
Steen
S
Nielsen
Kirsten Vang
KV
Nexø
Bjørn A
BA
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
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