Extension of the GOOD assay for genotyping single nucleotide polymorphisms by matrix-assisted laser desorption/ionization mass spectrometry.
Over the past years several methods using mass spectrometry for high-throughput genotyping of single nucleotide polymorphisms (SNPs) have been developed. Most of these procedures require stringent purification. Only the GOOD assay does not need any sample purification. Here, several new implementations of this assay are presented. The molecular biological procedure of the GOOD assays is based on the principle that the analysis of DNA by matrix-assisted laser desorption/ionization (MALDI) is strongly dependent on the charge state. A 100-fold increase in sensitivity can be achieved if the analyzed DNA product is conditioned by a chemical procedure termed 'charge-tagging'. The GOOD assay starts with a PCR; allele-specific DNA molecules are generated by extension of modified primers. These contain up to three phosphorothioates and optionally a quaternary ammonium charged group with ddNTPs or alpha-S-ddNTPs. Then the unmodified part of the primers is digested by phosphodiesterase II and the negative charges of the phosphorothioates are neutralized by an alkylation reaction resulting in charge-tagged DNA products. Through the use of a novel DNA polymerase for the primer extension, which preferably incorporates ddNTPs over dNTPs, an enzymatic degradation of residual dNTPs from the PCR is not required. Additionally, the unique property of charge-tag technology is demonstrated to detect specifically on the same sample allele-specific DNA products carrying a positive charge-tag in the positive ion mode while products carrying a negative charge-tag are analyzed in the negative ion mode. We also generated zwitterionic allele-specific products that were detectable with high sensitivity in positive ion mode. The findings of this study raise interesting questions about the ionization process of nucleic acids in MALDI. The new variations of the GOOD assay were applied to genotype SNPs of a candidate gene for cardiovascular disease.
Sauer
Sascha
S
Max-Planck-Institute for Molecular Genetics, Abteilung Lehrach, Ihnestrasse 73, 14195 Berlin-Dahlem, Germany.|Freie Universität Berlin, Fachbereich Biologie, Chemie, Pharmazie, Takustr. 3, 14195 Berlin-Dahlem, Germany.|Centre National de Génotypage, Bâtiment G2, 2 rue Gaston Crémieux, 91057 Evry Cedex, France.
Gut
Ivo G
IG
Centre National de Génotypage, Bâtiment G2, 2 rue Gaston Crémieux, 91057 Evry Cedex, France.
D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D005092:Exonucleases / Q000378:metabolism; D005838:Genotype; D006801:Humans; D008970:Molecular Weight; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D017931:DNA Primers; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases; C022650:spleen exonuclease