Background: Genetic diversity can help explain disease susceptibility and d
ifferential drug response. The most common type of variant is the single nu
cleotide polymorphism (SNP). We present a low-cost, high throughput assay f
or SNP genotyping.
Methods: The assay uses oligonucleotide probes covalently attached to fluor
escently encoded microspheres. These probes are hybridized directly to fluo
rescently labeled polymerase chain reaction (PCR) products and the results
are analyzed in a standard flow cytometer.
Results: The genotypes determined with our assay are in good agreement with
those determined by TaqMan. The range of G/C content for oligonucleotide p
robes mas 23.5-65% in the 17 bases surrounding the SNP. Further optimizatio
n of probe length and target concentration is shown to dramatically enhance
the assay performance for certain SNPs. Using microspheres which have uniq
ue fluorescent signatures, we performed a 32-plex assay where we simultaneo
usly determined the genotypes of eight different polymorphic genes.
Conclusions: We demonstrate, for the first time, the feasibility of multipl
exed genotyping with suspension arrays using direct hybridization analyses.
Our approach enables probes to be removed from or added to an array, entra
ncing flexibility over conventional chips. The ability to multiplex both th
e PCR preparation and the hybridization should enhance the throughput, cost
, and speed of the assay. Cytometry 10:102-108, 2000. (C) 2000 Wiley-Liss,