SNP genotyping using alkali cleavage of RNA/DNA chimeras and MALDI time-of-flight mass spectrometry.
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.
Centre National de Génotypage, Bâtiment G2 2 Rue Gaston Crémieux, 91057 Evry Cedex, France.
Florence
F
Mauger
Olivier
O
Jaunay
Valérie
V
Chamblain
Fred
F
Reichert
Keith
K
Bauer
Ivo G
IG
Gut
David H
DH
Gelfand
D000483:Alleles; D004247:DNA / Q000378:metabolism; D017931:DNA Primers; D003854:Deoxyribonucleotides / Q000378:metabolism; D005838:Genotype; D006878:Hydroxides; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D012313:RNA / Q000378:metabolism; D012265:Ribonucleotides / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D017931:DNA Primers; D003854:Deoxyribonucleotides; D006878:Hydroxides; D012265:Ribonucleotides; D012313:RNA; D004247:DNA