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SNP genotyping using microsphere-linked PNA and flow cytometric detection

Rockenbauer, Eszter LU ; Petersen, K; Vogel, U; Bolund, L; Kolvraa, S; Nielsen, K V and Nexo, B A (2005) In Cytometry Part A 64A(2). p.80-86
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.... (More)
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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
peptide nucleic acid, single nucleotide polymorphism, genotyping, flow cytometry, microspheres
in
Cytometry Part A
volume
64A
issue
2
pages
80 - 86
publisher
John Wiley & Sons
external identifiers
  • wos:000228153700004
  • pmid:15729710
  • scopus:17044398866
ISSN
1552-4930
DOI
10.1002/cyto.a.20123
language
English
LU publication?
yes
id
94bb1398-11b8-4cf7-bd90-c493354ffa8d (old id 246734)
date added to LUP
2007-10-05 12:45:51
date last changed
2017-01-01 05:16:40
@article{94bb1398-11b8-4cf7-bd90-c493354ffa8d,
  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.},
  author       = {Rockenbauer, Eszter and Petersen, K and Vogel, U and Bolund, L and Kolvraa, S and Nielsen, K V and Nexo, B A},
  issn         = {1552-4930},
  keyword      = {peptide nucleic acid,single nucleotide polymorphism,genotyping,flow cytometry,microspheres},
  language     = {eng},
  number       = {2},
  pages        = {80--86},
  publisher    = {John Wiley & Sons},
  series       = {Cytometry Part A},
  title        = {SNP genotyping using microsphere-linked PNA and flow cytometric detection},
  url          = {http://dx.doi.org/10.1002/cyto.a.20123},
  volume       = {64A},
  year         = {2005},
}