MALDI-TOF mass spectrometry for high throughput SNP detection: Application to type 2 
diabetes

Erdos, M., Braun, A., Mohlke, K., Silander, K., Ghosh, S., Collins, F.S. for the FUSION 
(Finland-US Investigation of NIDDM) study group.  NHGRI, NIH, Bethesda, MD, Sequenom
Inc., San Diego, CA

Matrix-Assisted Laser Desorption Ionization (MALDI) mass spectrometry has recently been 
introduced as a powerful analytical tool for the analysis of short DNA sequences by
mass.  Ions are generated by laser bombardment of DNA fragments embedded in a matrix of
3-hydroxypicolinic acid.  These large DNA ions are accelerated through a path towards
the detector measuring the time of flight (TOF) which is correlated with the mass of
the DNA fragment.  SNP analysis by MALDI-TOF is initiated by multiplex PCR amplification
of genomic DNA, using primers flanking the SNPs of interest.  A locus-specific primer
which abuts the variable nucleotide of each SNP is then used in a primer oligo base
extension reaction; primer extension is performed with DNA polymerase in the presence
of dideoxynucleotides that terminate the reactions at a specific length determined by
the allele present.  The extended products are then purified and subjected to mass
spectrometric analysis.  In a pilot study, we have designed and successfully implemented
the multiplex analysis by MALDI-TOF mass spectrometry of four genes with known
polymorphisms that have been implicated in diabetes. The variants tested are SNPs in
peroxisome proliferator-activated receptor gamma 2, insulin receptor substrate-1,
uncoupling protein-2 and glucokinase genes.  These genes are known to play a role in
insulin sensitivity, insulin receptor signaling, obesity, and/or glucose metabolism,
all traits which are associated with type 2 diabetes.  Genotypes in the pilot study
have been highly reproducible.  We are currently extending this analysis to 582
probands of diabetic sib-pairs and 232 controls consisting of elderly non-diabetic
Finnish subjects.  Comparison of SNP allele frequencies between diabetic probands and
controls should allow the identification or elimination of these candidate gene variants
for association with type 2 diabetes in the Finnish population.