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Related Experiment Videos

Fluorescent microsphere-based readout technology for multiplexed human single nucleotide polymorphism analysis and

F Ye1, M S Li, J D Taylor

  • 1Department of Genomic Sciences, Glaxo Wellcome Research and Development, Research Triangle Park, North Carolina 27709-3398, USA.

Human Mutation
|April 11, 2001
PubMed
Summary

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A new, rapid, and cost-effective method for single nucleotide polymorphism (SNP) genotyping combines allele-specific primer extension (ASPE) with flow cytometry. This high-throughput technique accurately genotypes human DNA and identifies bacterial species.

Area of Science:

  • Genetics and Genomics
  • Biotechnology
  • Molecular Biology

Background:

  • Large-scale human genotyping demands efficient, accurate, and automatable technologies.
  • Existing methods may lack cost-effectiveness or scalability for high-throughput applications.

Purpose of the Study:

  • To develop a rapid, cost-effective, and automatable method for single nucleotide polymorphism (SNP) genotyping.
  • To enable high-throughput multiplex SNP analysis using flow cytometry.
  • To adapt the assay for bacterial identification.

Main Methods:

  • Developed a single-tube allele-specific primer extension (ASPE) assay.
  • Utilized Luminex 100 flow cytometry for high-throughput analysis.
  • Employed ZipCode-tagged capture probes hybridized to ASPE products on microspheres.

Related Experiment Videos

  • Adapted the system for bacterial 16S rDNA identification using ASPE and single base chain extension (SBCE).

    • Main Results:

    • The microsphere-based ASPE assay demonstrated high accuracy, with 99.2% agreement with gel-based oligonucleotide ligation assay (OLA) for human SNP genotyping.
    • Successfully multiplexed 15 SNPs on human chromosome 12 from 96 patients.
    • The system effectively identified 17 bacterial species based on 16S rDNA variable sites, generating species- or genus-specific patterns consistent with DNA sequencing.

    Conclusions:

    • The developed ASPE-based flow cytometric assay is a robust, streamlined, and cost-effective solution for high-throughput SNP genotyping.
    • This technique offers flexibility for multiplex analysis of various allelic variants.
    • The microsphere-based assay is adaptable for microbial identification, broadening its application in molecular diagnostics and research.