High-throughput multiplex SNP genotyping with MALDI-TOF mass spectrometry: Practice, problems and promise
Article first published online: 2 APR 2001
Copyright © 2001 Wiley-Liss, Inc.
Special Issue: SNP 2000: Third International Meeting on Single Nucleotide Polymorphism and Complex Genome Analysis
Volume 17, Issue 4, pages 296–304, April 2001
How to Cite
Bray, M. S., Boerwinkle, E. and Doris, P. A. (2001), High-throughput multiplex SNP genotyping with MALDI-TOF mass spectrometry: Practice, problems and promise. Hum. Mutat., 17: 296–304. doi: 10.1002/humu.27
- Issue published online: 2 APR 2001
- Article first published online: 2 APR 2001
- Manuscript Accepted: 12 JAN 2001
- Manuscript Received: 8 NOV 2000
- CDC. Grant Number: UR6/CCU617218-01
- NIH. Grant Numbers: HL54481, HL54526, HL54457, HL54464, DDK45538
- mass spectrometry;
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. © 2001 Wiley-Liss, Inc.