Application of the C4'-alkylated deoxyribose primer system (CAPS) in allele-specific real-time PCR for increased selectivity in discrimination of single nucleotide sequence variants

Tews, Björn; Wilhelm, Jochen; Summerer, Daniel; Strerath, Michael; Marx, Andreas; Friedhoff, Peter; Pingoud, Alfred; Hahn, Meinhard

Application of the C4'-alkylated deoxyribose primer system (CAPS) in allele-specific real-time PCR for increased selectivity in discrimination of single nucleotide sequence variants

Dateien

Summerer.pdfGröße: 26.64 MBDownloads: 503

Datum

2003

Autor:innen

Tews, Björn

Wilhelm, Jochen

Summerer, Daniel

Strerath, Michael

Marx, Andreas

Friedhoff, Peter

Pingoud, Alfred

Hahn, Meinhard

Open Access-Veröffentlichung

Open Access Green

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Zukunftskolleg

Publikationstyp

Zeitschriftenartikel

Publikationsstatus

Published

Erschienen in

Biological Chemistry. 2003, 384(10-11), pp. 1533-1541. ISSN 1431-6730. Available under: doi: 10.1515/BC.2003.170

Zusammenfassung

This study describes a quantitative real-time PCR-based approach for discrimination of single nucleotide sequence variants, called CAPS (C4 /alkylated primer system). To increase the discrimination potential of DNA polymerases against competing sequence variants of single nucleotides, 3'-terminally modified primers were designed carrying a methyl residue bound to the C4' of the thymidylate deoxyribose. In a model sequence system positional dependencies of modified thymidylate (at -1, -2, -3) were tested for their influence on discrimination. Highest discrimination factors were obtained with the modification at the ultimate 3'-position. In a comparison between Taq and Pwo DNA polymerases, substantial better results were obtained by Taq DNA polymerase. In contrast to conventional PCR methods for discrimination of sequence variants, achieving a maximum discrimination potential of about 20, CAPS is capable of obtaining sequence-specific amplifications of a desired target among discriminated templates with a dynamic range of 1:100. Therefore, CAPS is a method able to quantitatively discriminate two sequence variants only differing in a single base (e.g., SNP alleles or point mutations). The range of applications of this easy to perform, fast and reliable technique reaches from medical diagnostics, transplantation medicine, molecular and cell biology to human genetics. Targeting of SNPs assures a universal exertion of this method, since these markers are gender-independent, highly abundant and ubiquitous.

Fachgebiet (DDC)

540 Chemie

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ISO 690

TEWS, Björn, Jochen WILHELM, Daniel SUMMERER, Michael STRERATH, Andreas MARX, Peter FRIEDHOFF, Alfred PINGOUD, Meinhard HAHN, 2003. Application of the C4'-alkylated deoxyribose primer system (CAPS) in allele-specific real-time PCR for increased selectivity in discrimination of single nucleotide sequence variants. In: Biological Chemistry. 2003, 384(10-11), pp. 1533-1541. ISSN 1431-6730. Available under: doi: 10.1515/BC.2003.170

BibTex

@article{Tews2003-10Appli-422,
  year={2003},
  doi={10.1515/BC.2003.170},
  title={Application of the C4'-alkylated deoxyribose primer system (CAPS) in allele-specific real-time PCR for increased selectivity in discrimination of single nucleotide sequence variants},
  number={10-11},
  volume={384},
  issn={1431-6730},
  journal={Biological Chemistry},
  pages={1533--1541},
  author={Tews, Björn and Wilhelm, Jochen and Summerer, Daniel and Strerath, Michael and Marx, Andreas and Friedhoff, Peter and Pingoud, Alfred and Hahn, Meinhard},
  note={Article Number: 1533-41}
}

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