SNP genotyping using alkali cleavage of RNA/DNA chimeras and MALDI time-of-flight mass spectrometry
Identifieur interne : 001616 ( Main/Exploration ); précédent : 001615; suivant : 001617SNP genotyping using alkali cleavage of RNA/DNA chimeras and MALDI time-of-flight mass spectrometry
Auteurs : Florence Mauger ; Olivier Jaunay ; Vale Rie Chamblain ; Fred Reichert [États-Unis] ; Keith Bauer [États-Unis] ; Ivo G. Gut ; David H. Gelfand [États-Unis]Source :
- Nucleic Acids Research [ 0305-1048 ] ; 2006.
Abstract
Single nucleotide polymorphisms (SNPs) are now widely used for many DNA analysis applications such as linkage disequilibrium mapping, pharmacogenomics and traceability. Many methods for SNP genotyping exist with diverse strategies for allele-distinction. Mass spectrometers are used most commonly in conjunction with primer extension procedures with allele-specific termination. Here we present a novel concept for allele-preparation for SNP genotyping. Primer extension is carried out with an extension primer positioned immediately upstream of the SNP that is to be genotyped, a complete set of four ribonucleotides and a ribonucleotide incorporating DNA polymerase. The allele-extension products are then treated with alkali, which results in the cleavage immediately after the first added ribonucleotide. In addition, to obtain fragments easily detectable by mass spectrometry, we have included a ribonucleotide in the primer usually at the fourth nucleotide from the 3′ terminus. The method was tested on four SNPs each with a different combination of nucleotides. The advantage over other mass spectrometry-based SNP genotyping assays is that this one only requires a PCR, a primer extension reaction with a universal extension mix and an inexpensive facile cleavage reaction, which makes it overall very cost effective and easy in handling.
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DOI: 10.1093/nar/gnj021
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Many methods for SNP genotyping exist with diverse strategies for allele-distinction. Mass spectrometers are used most commonly in conjunction with primer extension procedures with allele-specific termination. Here we present a novel concept for allele-preparation for SNP genotyping. Primer extension is carried out with an extension primer positioned immediately upstream of the SNP that is to be genotyped, a complete set of four ribonucleotides and a ribonucleotide incorporating DNA polymerase. The allele-extension products are then treated with alkali, which results in the cleavage immediately after the first added ribonucleotide. In addition, to obtain fragments easily detectable by mass spectrometry, we have included a ribonucleotide in the primer usually at the fourth nucleotide from the 3′ terminus. The method was tested on four SNPs each with a different combination of nucleotides. 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Gut</name><name sortKey="Jaunay, Olivier" sort="Jaunay, Olivier" uniqKey="Jaunay O" first="Olivier" last="Jaunay">Olivier Jaunay</name><name sortKey="Mauger, Florence" sort="Mauger, Florence" uniqKey="Mauger F" first="Florence" last="Mauger">Florence Mauger</name></noCountry><country name="États-Unis"><region name="Californie"><name sortKey="Reichert, Fred" sort="Reichert, Fred" uniqKey="Reichert F" first="Fred" last="Reichert">Fred Reichert</name></region><name sortKey="Bauer, Keith" sort="Bauer, Keith" uniqKey="Bauer K" first="Keith" last="Bauer">Keith Bauer</name><name sortKey="Gelfand, David H" sort="Gelfand, David H" uniqKey="Gelfand D" first="David H." last="Gelfand">David H. Gelfand</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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