Open AccessSuspension arrays for high throughput, multiplexed single nucleotide polymorphism genotyping
Author(s) -
Armstrong Barbara,
Stewart Michael,
Mazumder Abhijit
Publication year - 2000
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/(sici)1097-0320(20000601)40:2<102::aid-cyto3>3.0.co;2-4
Subject(s) - genotyping , multiplex , taqman , snp genotyping , molecular inversion probe , oligonucleotide , biology , microbiology and biotechnology , single nucleotide polymorphism , multiplex polymerase chain reaction , genotype , polymerase chain reaction , computational biology , genetics , gene
Background Genetic diversity can help explain disease susceptibility and differential drug response. The most common type of variant is the single nucleotide polymorphism (SNP). We present a low‐cost, high throughput assay for SNP genotyping. Methods The assay uses oligonucleotide probes covalently attached to fluorescently encoded microspheres. These probes are hybridized directly to fluorescently 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 probes was 23.5–65% in the 17 bases surrounding the SNP. Further optimization of probe length and target concentration is shown to dramatically enhance the assay performance for certain SNPs. Using microspheres which have unique fluorescent signatures, we performed a 32‐plex assay where we simultaneously determined the genotypes of eight different polymorphic genes. Conclusions We demonstrate, for the first time, the feasibility of multiplexed genotyping with suspension arrays using direct hybridization analyses. Our approach enables probes to be removed from or added to an array, enhancing flexibility over conventional chips. The ability to multiplex both the PCR preparation and the hybridization should enhance the throughput, cost, and speed of the assay. Cytometry 40:102–108, 2000. © 2000 Wiley‐Liss, Inc.