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Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry
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Multiplexed SNP genotyping using primer single-base extension (SBE) and microsphere arrays.

Alina Deshpande1, Yolanda Valdez, John P Nolan

  • 1Los Alamos National Laboratory, Los Alamos, New Mexico, USA.

Current Protocols in Cytometry
|September 5, 2008
PubMed
Summary

This study details a method for multiplexed single-nucleotide polymorphism (SNP) genotyping using primer extension on microspheres and flow cytometry. This technique efficiently analyzes genetic variations in numerous samples for research and clinical applications.

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Area of Science:

  • Genetics
  • Molecular Biology
  • Biotechnology

Background:

  • Single-nucleotide polymorphisms (SNPs) are common genetic variations influencing phenotypes, disease susceptibility, and pathogen resistance.
  • SNPs serve as crucial markers for DNA allelic segments, making them valuable in basic and clinical research.
  • Efficient and scalable genotyping methods are essential for studying genetic variations.

Purpose of the Study:

  • To present detailed protocols for multiplexed SNP genotyping.
  • To adapt primer single-base extension (SBE) technology for use with microspheres and flow cytometry.
  • To provide a method suitable for typing a modest number of SNPs in a large sample cohort.

Main Methods:

  • Multiplexed SNP genotyping using primer single-base extension (SBE).
  • Adaptation of SBE to microsphere-based assays for high-throughput analysis.
  • Utilizing flow cytometry for detection and measurement of extended primers captured on addressable microspheres.

Main Results:

  • The protocol enables the extension of genotyping primers by a single, labeled dideoxyribonucleotide.
  • This labeled nucleotide directly reveals the base at the polymorphic site on the template DNA strand.
  • Extended primers are captured on microspheres with specific oligonucleotide addresses for subsequent flow cytometric analysis.

Conclusions:

  • The described method provides a robust approach for multiplexed SNP genotyping.
  • The combination of SBE, microspheres, and flow cytometry is effective for analyzing genetic variations in large sample sets.
  • This technique facilitates the use of SNPs as tools in both fundamental biological research and clinical diagnostics.