Facile method for automated genotyping of single nucleotide polymorphisms by mass spectrometry.
In the future, analysis of single nucleotide polymorphisms (SNPs) should become a powerful tool for many genetic applications in areas such as association studies, pharmacogenetics and traceability in the agro-alimentary sector. A number of technologies have been developed for high-throughput genotyping of SNPs. Here we present the simplified GOOD assay for SNP genotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI). The simplified GOOD assay is a single-tube, purification-free, three-step procedure consisting of PCR, primer extension and phosphodiesterase II digestion followed by mass spectrometric analysis. Due to the application of charge-tag technology, no sample purification is required prior to the otherwise very impurity-sensitive MALDI analysis. The use of methylphosphonate containing primers and ddNTPs or alpha-S-ddNTPs together with a novel DNA polymerase derived from Thermotoga maritima for primer extension allow the fluent preparation of negatively charge-tagged, allele-specific products. A key feature of this polymerase is its preference for ddNTPs and alpha-S-ddNTPs over dNTPs. The simplified GOOD assay was run with automatic liquid handling at the lowest manageable volumes, automatic data acquisition and interpretation. We applied this novel procedure to genotyping SNPs of candidate genes for hypertension and cardiovascular disease.
Sauer
Sascha
S
Freie Universität Berlin, Fachbereich Biologie, Chemie, Pharmazie, Takustrasse 3, 14195 Berlin-Dahlem, Germany.
Gelfand
David H
DH
Boussicault
Francis
F
Bauer
Keith
K
Reichert
Fred
F
Gut
Ivo G
IG
D001331:Automation; D002318:Cardiovascular Diseases / Q000235:genetics; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D005838:Genotype; D006801:Humans; D006973:Hypertension / Q000235:genetics; D009943:Organophosphorus Compounds / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D017931:DNA Primers; D009943:Organophosphorus Compounds; D018343:Receptors, Adrenergic, beta-2; C032627:methylphosphonic acid; D004259:DNA-Directed DNA Polymerase