A novel procedure for efficient genotyping of single nucleotide polymorphisms.
Due to the surge in interest in using single nucleotide polymorphisms (SNPs) for genotyping a facile and affordable method for this is an absolute necessity. Here we introduce a procedure that combines an easily automatable single tube sample preparation with an efficient high throughput mass spectrometric analysis technique. Known point mutations or single nucleotide polymorphisms are easily analysed by this procedure. It starts with PCR amplification of a short stretch of genomic DNA, for example an exon of a gene containing a SNP. By shrimp alkaline phosphatase digest residual dNTPs are destroyed. Allele-specific products are generated using a special primer, a conditioned set of alpha-S-dNTPs and alpha-S-ddNTPs and a fresh DNA polymerase in a primer extension reaction. Unmodified DNA is removed by 5'-phospho-diesterase digestion and the modified products are alkylated to increase the detection sensitivity in the mass spectrometric analysis. All steps of the preparation are simple additions of solutions and incubations. The procedure operates at the lowest practical sample volumes and in contrast to other genotyping protocols with mass spectrometric detection requires no purification. This reduces the cost and makes it easy to implement. Here it is demonstrated in a version using positive ion detection on described mutations in exon 17 of the amyloid precursor protein gene and in a version using negative ion detection on three SNPs of the granulocyte-macrophage colony stimulating factor gene. Preparation and analysis of SNPs is shown separately and simultaneously, thus demonstrating the multiplexibility of this genotyping procedure. The preparation protocol for genotyping is adapted to the conditions used for the SNP discovery method by denaturing HPLC, thus demonstrating a facile link between protocols for SNP discovery and SNP genotyping. Results corresponded unanimously with the control sequencing. The procedure is useful for high throughput genotyping as it is required for gene identification and pharmacogenomics where large numbers of DNA samples have to be analysed. We have named this procedure the 'GOOD Assay' for SNP analysis.
Sauer
S
S
Max-Planck-Institute for Molecular Genetics, Abteilung Lehrach, Ihnestrasse 73, 14195 Berlin-Dahlem, Germany.
Lechner
D
D
Berlin
K
K
Lehrach
H
H
Escary
J L
JL
Fox
N
N
Gut
I G
IG
D016564:Amyloid beta-Protein Precursor / Q000235:genetics*; D003062:Codon; D004252:DNA Mutational Analysis; D005821:Genetic Techniques; D005838:Genotype; D016179:Granulocyte Colony-Stimulating Factor / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D016564:Amyloid beta-Protein Precursor; D003062:Codon; D016179:Granulocyte Colony-Stimulating Factor