High-Throughput Genotyping with Single Nucleotide Polymorphisms (original) (raw)

  1. Koustubh Ranade1,10,11,12,
  2. Mau-Song Chang2,
  3. Chih-Tai Ting3,
  4. Dee Pei4,
  5. Chin-Fu Hsiao5,
  6. Michael Olivier1,
  7. Robert Pesich1,
  8. Joan Hebert1,
  9. Yii-Der I. Chen6,
  10. Victor J. Dzau7,
  11. David Curb8,
  12. Richard Olshen9,
  13. Neil Risch1,
  14. David R. Cox1, and
  15. David Botstein1,11,13
  16. 1Department of Genetics, Stanford University School of Medicine, Stanford, California 94305–5120, USA; 2Department of Medicine, Veterans General Hospital, Taipei, Taiwan; 3Department of Medicine, Veterans General Hospital, Taichung, Taiwan; 4Department of Endocrinology and Metabolism, Tri-Service General Hospital, Taipei, Taiwan;5National Health Research Institute, Taipei, Taiwan;6Division of Endocrinology, Stanford University School of Medicine, Stanford, California 94305, USA; 7Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA; 8Hawaii Center for Health Research, Honolulu, Hawaii 96813, USA; 9Health Research and Policy, Stanford University School of Medicine, Stanford, California 94305, USA

Abstract

To make large-scale association studies a reality, automated high-throughput methods for genotyping with single-nucleotide polymorphisms (SNPs) are needed. We describe PCR conditions that permit the use of the TaqMan or 5′ nuclease allelic discrimination assay for typing large numbers of individuals with any SNP and computational methods that allow genotypes to be assigned automatically. To demonstrate the utility of these methods, we typed >1600 individuals for a G-to-T transversion that results in a glutamate-to-aspartate substitution at position 298 in the endothelial nitric oxide synthase gene, and a G/C polymorphism (newly identified in our laboratory) in intron 8 of the 11–β hydroxylase gene. The genotyping method is accurate—we estimate an error rate of fewer than 1 in 2000 genotypes, rapid—with five 96-well PCR machines, one fluorescent reader, and no automated pipetting, over one thousand genotypes can be generated by one person in one day, and flexible—a new SNP can be tested for association in less than one week. Indeed, large-scale genotyping has been accomplished for 23 other SNPs in 13 different genes using this method. In addition, we identified three “pseudo-SNPs” (WIAF1161, WIAF2566, and WIAF335) that are probably a result of duplication.

Footnotes