Multiplexed SNP Genotyping Using Primer Single‐Base Extension (SBE) and Microsphere Arrays

Alina Deshpande1, Yolanda Valdez1, John P. Nolan2

1 Los Alamos National Laboratory, Los Alamos, New Mexico, 2 La Jolla Bioengineering Institute, La Jolla, California
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 13.4
DOI:  10.1002/0471142956.cy1304s34
Online Posting Date:  November, 2005
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Abstract

Single‐nucleotide polymorphisms (SNPs), genome sites with single‐base sequence differences between individual chromosomes, are the most common type of genetic variation. Single‐base changes can result in disease phenotypes, confer susceptibility or resistance to toxins or pathogens, or serve as markers for distinct allelic segments of DNA, making SNPs an important emerging tool in both basic and clinical research. This unit presents detailed protocols for multiplexed SNP genotyping using primer single‐base extension (SBE) adapted to microspheres and flow cytometry. The methods described are best suited for typing a modest number of SNPs in a large number of samples. The Basic Protocol describes extension of the genotyping primers by one nucleotide, a labeled dideoxyribonucleotide that reveals the nucleotide base at that position on the template strand. The extended primers are then captured onto microspheres bearing an oligonucleotide “address” that is the reverse complement of a sequence on the 5′ end of the genotyping primers and subsequently measured using flow cytometry.

Keywords: genotyping; single‐base extension; microsphere arrays; primers; address‐tagged beads

     
 
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Table of Contents

  • Basic Protocol 1: Single‐Base Primer Extension
  • Support Protocol 1: Assay Primer and Address Tag Design
  • Support Protocol 2: PCR Product Cleanup
  • Support Protocol 3: Preparation of Address‐Tagged Microspheres
  • Support Protocol 4: Data Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Single‐Base Primer Extension

  Materials
  • Genotyping samples (amplified, cleaned PCR products from samples of interest; see protocol 3; see protocol 2 for primer design)
  • Genotyping controls (amplified, cleaned PCR products from genotyping samples with known genotypes; see protocol 3; see protocol 2 for primer design)
  • SBE genotyping reaction mixes (see recipe)
  • Address‐tagged microsphere array (see protocol 4)
  • Wash buffer (see recipe)
  • 33 nM streptavidin‐phycoerythrin (SA‐PE; Molecular Probes)
  • 96‐well plate (or PCR strip tubes)
  • Thermal cycler
  • Centrifuge with 96‐well plate– or PCR strip tube–adapted rotor
  • Flow cytometer

Support Protocol 1: Assay Primer and Address Tag Design

  Materials
  • PCR product (amplified target DNA)
  • Shrimp alkaline phosphatase (SAP; USB)
  • Exonuclease I (Exo I; USB)
  • 100 mM Tris⋅Cl, pH 8
  • 25 mM MgCl 2
  • 37° and 72°C water baths or heating blocks

Support Protocol 2: PCR Product Cleanup

  Materials
  • 50 mM 2‐(N‐morpholino)ethanesulfonic acid, pH 6.5 (MES; Sigma)
  • 100 µM amino‐modified address‐tag oligonucleotides (see protocol 2)
  • Optically encoded microspheres (see above; resuspended in 50 mM MES, pH 6.5)
  • 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide hydrochloride (EDC; Pierce)
  • N‐hydroxysuccinimide (NHS; Aldrich)
  • 50 mM MES, pH 6.5, with 0.05% (v/v) Tween 20
  • NaCl stock solution
  • Centrifuge tubes, siliconized (Fisher)
  • Platform shaker
  • Centrifuge
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Figures

Videos

Literature Cited

Literature Cited
   Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (eds.) 2005. Current Protocols in Molecular Biology, Chapter 2. John Wiley & Sons, Hoboken, N.J.
   Brookes, A.J. 1999. The essence of SNPs. Gene 234:177‐186.
   Cai, H., White, P.S., Torney, D.C., Deshpande, A., Wang, Z., Keller, R.A., Marrone, B.L., and Nolan, J.P. 2000. Flow cytometry based minisequencing: A new platform for high throughput single nucleotide polymorphism analysis. Genomics 66:135‐143.
   Chen, J., Iannone, M.A., Li, M.S., Taylor, J.D., Rivers, P., Nelsen, A.J., Slentz‐Kesler, K.A., Roses, A., and Weiner, M.P. 2000. A microsphere‐based assay for multiplexed single nucleotide polymorphism analysis using single base chain extension. Genome Res. 10:549‐557.
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   Kaderali, L., Deshpande, A., Nolan, J.P., and White, P.S. 2003. Primer design for multiplexed genotyping. Nucleic Acids Res. 31:1796‐1802.
   Kramer, M.F. and Coen, D.M. 2001. Enzymatic amplification of DNA by PCR: standard procedures and optimization. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 15.1.1‐15.1.14. John Wiley & Sons, Hoboken, N.J.
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   Syvanen, A.C. 1999. From gels to chips: “minisequencing” primer extension for analysis of point mutations and single nucleotide polymorphisms. Hum. Mutat. 13:1–10.
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