Detection of mitochondrial single nucleotide polymorphisms using a primer elongation reaction on oligonucleotide microarrays
Författare
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.
Publiceringsår
2001
Språk
Engelska
Sidor
36-36
Publikation/Tidskrift/Serie
Nucleic Acids Research
Volym
29
Issue
7
Dokumenttyp
Artikel i tidskrift
Förlag
Oxford University Press
Ämne
- Cell and Molecular Biology
Status
Published
ISBN/ISSN/Övrigt
- ISSN: 1362-4962