High-throughput multiplex SNP genotyping with MALDI-TOF mass spectrometry:Practice, problems and promise

Single nucleotide polymorphisms (SNPs) are currently being identified and m apped at a remarkable pace, providing a rich genetic resource with vast pot ential for disease gene discovery, pharmacogenetics, and understanding the origins of modern humans. High-throughput, cost effective genotyping method s 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 deso rption/ionization time-of-flight mass spectrometry (MALDI-TOF) in our labor atory as a tool for differentiating genotypes based on the mass of the vari ant DNA sequence, and have utilized this method for production scale SNP ge notyping. We have combined a 4 mul PCR amplification reaction using 3 ng of genomic DNA with a secondary enzymatic reaction (mini-sequencing) containi ng oligonucleotide primers that anneal immediately upstream of the polymorp hic site, dideoxynucleotides, and a thermostable polymerase used to extend the PCR product by a single base pair. Mass spectrometry (MS) analysis of m ini sequencing reactions was performed using a MALDI-TOF instrument (Voyage r-DF, 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 wi th 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 detec ted in a single reaction, the MS technique provides a cost-effective and ef ficient method for high throughput genotyping, Hum Mutat 17:296-304, 2001, (C) 2001 Wiley-Liss, Inc.