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

Hum Mutat. 2001 Apr;17(4):296-304. doi: 10.1002/humu.27.


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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alleles
  • Costs and Cost Analysis
  • DNA Mutational Analysis / economics
  • DNA Mutational Analysis / methods
  • DNA Primers / genetics
  • Gene Frequency / genetics
  • Genetic Testing / economics
  • Genetic Testing / methods*
  • Genotype
  • Humans
  • Molecular Sequence Data
  • Mutation, Missense / genetics
  • Polymerase Chain Reaction
  • Polymorphism, Single Nucleotide / genetics*
  • Sensitivity and Specificity
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization*


  • DNA Primers

Associated data

  • GENBANK/M76722
  • GENBANK/X15323
  • GENBANK/Z11162