title: High-throughput multiplex SNP genotyping with MALDI-TOF mass spectrometry: practice, problems and promise.
abstract: Single nucleotide polymorphisms (SNPs) are currently being identified and mapped at a remarkable pace, providing a rich genetic resource with vast potential for disease gene discovery, pharmacogenetics, and understanding the origins of modern humans. High-throughput, cost effective genotyping methods are essential in order to make the most advantageous and immediate use of these SNP data. We have incorporated the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) in our laboratory as a tool for differentiating genotypes based on the mass of the variant DNA sequence, and have utilized this method for production scale SNP genotyping. We have combined a 4 microl PCR amplification reaction using 3 ng of genomic DNA with a secondary enzymatic reaction (mini-sequencing) containing oligonucleotide primers that anneal immediately upstream of the polymorphic site, dideoxynucleotides, and a thermostable polymerase used to extend the PCR product by a single base pair. Mass spectrometry (MS) analysis of mini-sequencing reactions was performed using a MALDI-TOF instrument (Voyager-DE, Perseptive Biosystems, Framingham, MA). We performed both single and multiplex PCR and mini-sequencing reactions, and genotyped seven different variant sites in a random sample of 989 individuals. Genotypes generated with MS methods were compared with genotypes produced using a 5' exonuclease fluorescence-based assay (Taqman, Applied Biosystems, Foster City, CA) and a gel-based genotyping protocol. Because multiple polymorphisms can be detected in a single reaction, the MS technique provides a cost-effective and efficient method for high-throughput genotyping.
authors: Human Genetics Center, University of Texas Health Science Center at Houston, Houston, Texas, 77255, USA. molly.s.bray@uth.tmc.edu
authors: M S
authors: MS
authors: Bray
authors: E
authors: E
authors: Boerwinkle
authors: P A
authors: PA
authors: Doris
mesh_terms: D000483:Alleles; D003365:Costs and Cost Analysis; D004252:DNA Mutational Analysis / Q000191:economics / Q000379:methods; D017931:DNA Primers / Q000235:genetics; D005787:Gene Frequency / Q000235:genetics; D005820:Genetic Testing / Q000191:economics / Q000379:methods*; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D020125:Mutation, Missense / Q000235:genetics; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D017931:DNA Primers