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

Erdogan, Fikret; Kirchner, Roland; Mann, Wolfgang; Ropers, Hans-Hilger and Nuber, Ulrike LU (2001) In Nucleic Acids Research 29(7). p.36-36
Abstract
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... (More)
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. (Less)
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author
publishing date
type
Contribution to journal
publication status
published
subject
in
Nucleic Acids Research
volume
29
issue
7
pages
36 - 36
publisher
Oxford University Press
external identifiers
  • pmid:11266571
  • scopus:0035315802
ISSN
1362-4962
language
English
LU publication?
no
id
13925d70-f249-4959-a5ea-3cd8840fc7ac (old id 1122952)
alternative location
http://www.ncbi.nlm.nih.gov/pubmed/11266571
date added to LUP
2008-06-27 09:01:03
date last changed
2018-06-17 03:56:18
@article{13925d70-f249-4959-a5ea-3cd8840fc7ac,
  abstract     = {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.},
  author       = {Erdogan, Fikret and Kirchner, Roland and Mann, Wolfgang and Ropers, Hans-Hilger and Nuber, Ulrike},
  issn         = {1362-4962},
  language     = {eng},
  number       = {7},
  pages        = {36--36},
  publisher    = {Oxford University Press},
  series       = {Nucleic Acids Research},
  title        = {Detection of mitochondrial single nucleotide polymorphisms using a primer elongation reaction on oligonucleotide microarrays},
  volume       = {29},
  year         = {2001},
}