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Detection of mitochondrial single nucleotide polymorphisms using a primer elongation reaction on oligonucleotide microarrays

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Author

  • Fikret Erdogan
  • Roland Kirchner
  • Wolfgang Mann
  • Hans-Hilger Ropers
  • Ulrike Nuber

Summary, in English

We have developed a novel allele-specific primer elongation protocol using a DNA polymerase on oligonucleotide chips. Oligonucleotide primers carrying polymorphic sites at their free 3'end were covalently bound to glass slides. The generation of single-stranded targets of genomic DNA containing single nuclotide polymorphisms (SNPs) to be typed was achieved by an asymmetric PCR reaction or exonuclease treatment of phosphothioate (PTO)-modified PCR products. In the presence of DNA polymerase and all four dNTPs, with Cy3-dUTP replacing dTTP, allele-specific extension of the immobilized primers took place along a stretch of target DNA sequence. The yield of elongated products was increased by repeated reaction cycles. We performed multiplexed assays with many small DNA targets, or used single targets of up to 4.4 kb mitochondrial DNA (mtDNA) sequence to detect multiple SNPs in one reaction. The latter approach greatly simplifies preamplification of SNP-containing regions, thereby providing a framework for typing hundreds of mtDNA polymorphisms.

Publishing year

2001

Language

English

Pages

36-36

Publication/Series

Nucleic Acids Research

Volume

29

Issue

7

Document type

Journal article

Publisher

Oxford University Press

Topic

  • Cell and Molecular Biology

Status

Published

ISBN/ISSN/Other

  • ISSN: 1362-4962