Molly S. Bray

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 μl 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. Hum Mutat 17:296–304, 2001.