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Multiplexed single nucleotide polymorphism genotyping by oligonucleotide ligation and flow cytometry.
M A Iannone1, J D Taylor, J Chen
1Department of Molecular Sciences, Glaxo Wellcome Research Laboratories, Research Triangle Park, NC 27709-3398, USA. mai49583@glaxowellcome.com
Cytometry
|February 19, 2000
Summary
This study introduces a fast, high-throughput method for single nucleotide polymorphism (SNP) genotyping using oligonucleotide ligation assay (OLA) and flow cytometry. The novel approach accurately analyzes multiple SNPs simultaneously in a single tube.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Developed a rapid, high-throughput method for single nucleotide polymorphism (SNP) genotyping.
- Employs oligonucleotide ligation assay (OLA) and flow cytometric analysis of fluorescent microspheres.
Purpose of the Study:
- To present a novel method for multiplexed SNP genotyping.
- To demonstrate the accuracy and speed of the OLA-based microsphere assay.
Main Methods:
- Utilizes OLA to add a reporter sequence to capture probes.
- Capture probes hybridize to PCR-amplified genomic targets and microsphere-coupled DNA (ZipCodes).
- Flow cytometry analyzes microsphere fluorescence for SNP identification in a multiplexed format.
Main Results:
- Successfully demonstrated multiplexed genotyping of nine SNP markers near the ApoE locus in seven CEPH DNA samples.
- Microsphere-based SNP analysis showed complete agreement with genotyping by sequencing.
- Validated the accuracy and efficiency of the developed methodology.
Conclusions:
- Multiplexed SNP genotyping using OLA and flow cytometric analysis of fluorescent microspheres is an accurate and rapid method.
- This technique offers a powerful tool for high-throughput SNP analysis.
- The method is suitable for various genetic studies requiring efficient SNP genotyping.