Multiplexed single nucleotide polymorphism genotyping by oligonucleotide ligation and flow cytometry

Cytometry. 2000 Feb 1;39(2):131-40.

Abstract

Background: We have developed a rapid, high throughput method for single nucleotide polymorphism (SNP) genotyping that employs an oligonucleotide ligation assay (OLA) and flow cytometric analysis of fluorescent microspheres.

Methods: A fluoresceinated oligonucleotide reporter sequence is added to a "capture" probe by OLA. Capture probes are designed to hybridize both to genomic "targets" amplified by polymerase chain reaction and to a separate complementary DNA sequence that has been coupled to a microsphere. These sequences on the capture probes are called "ZipCodes". The OLA-modified capture probes are hybridized to ZipCode complement-coupled microspheres. The use of microspheres with different ratios of red and orange fluorescence makes a multiplexed format possible where many SNPs may be analyzed in a single tube. Flow cytometric analysis of the microspheres simultaneously identifies both the microsphere type and the fluorescent green signal associated with the SNP genotype.

Results: Application of this methodology is demonstrated by the multiplexed genotyping of seven CEPH DNA samples for nine SNP markers located near the ApoE locus on chromosome 19. The microsphere-based SNP analysis agreed with genotyping by sequencing in all cases.

Conclusions: Multiplexed SNP genotyping by OLA with flow cytometric analysis of fluorescent microspheres is an accurate and rapid method for the analysis of SNPs.

MeSH terms

  • Chromosomes, Human, Pair 19
  • DNA / analysis
  • Flow Cytometry / methods*
  • Fluoresceins
  • Fluorescent Dyes
  • Genetic Markers
  • Genome, Human
  • Genotype
  • Humans
  • Microspheres
  • Nucleic Acid Hybridization
  • Oligonucleotides / chemistry*
  • Polymorphism, Single Nucleotide / genetics*

Substances

  • Fluoresceins
  • Fluorescent Dyes
  • Genetic Markers
  • Oligonucleotides
  • DNA