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Nucleic Acids Research, 2001, Vol. 29, No. 7 e36
© 2001 Oxford University Press

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

Fikret Erdogan1,2, Roland Kirchner1, Wolfgang Mann1, Hans-Hilger Ropers1 and Ulrike A. Nuber1,*

1Max-Planck Institute for Molecular Genetics, Ihnestrasse 73, 14195 Berlin, Germany and 2Freie Universität Berlin, Fachbereich Chemie, Biologie, Pharmazie, Takustrasse 3, 14195 Berlin, Germany

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.

* To whom correspondence should be addressed. Tel: +49 30 8413 1243; Fax: +49 30 8413 1383; Email: nuber{at}molgen.mpg.de Present address: Roland Kirchner and Wolfgang Mann, MWG Biotech AG, Anzinger Strasse 7, 85560 Ebersberg, Germany


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