High‐Throughput Genotyping with Primer Extension Fluorescent Polarization Detection

Pui‐Yan Kwok1

1 University of California, San Francisco, California
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 2.11
DOI:  10.1002/0471142905.hg0211s39
Online Posting Date:  February, 2004
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Abstract

The primer extension assay with fluorescence polarization (FP) detection is a versatile assay for single nucleotide polymorphism (SNP) genotyping. With proper design, this homogeneous assay works under universal conditions. Fluorescence polarization occurs when a fluorescent dye is excited by planeā€polarized light and is detected if the fluorescent dye is part of a large molecule. In assays where small fluorescent molecules are turned into large fluorescent products, FP provides a simple way to determine if the reaction has occurred without the need for separation or purification of the reaction mixture. In the primer extension assay, DNA polymerase incorporates the complementary, allelic nucleotide onto the SNP primer designed to anneal to the target DNA one base upstream of the polymorphic site. When a fluorescently labeled nucleotide is incorporated, high FP is observed for that dye and the genotype of the DNA sample is determined.

Keywords: Single nucleotide polymorphisms; fluorescence polarization; DNA sequence variation; homogeneous assays; single base extension; primer extension

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Primer Extension Assay with Single‐Plex PCR
  • Basic Protocol 2: Primer Extension Assay with 4‐Plex PCR
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Primer Extension Assay with Single‐Plex PCR

  Materials
  • 0.8 ng/µl genomic DNA of interest
  • 10× PCR buffer: 200 mM Tris·Cl, pH 8.4 ( appendix 2D)/500 mM KCl (may be purchased from Invitrogen)
  • 2.5 mM 4dNTP mix: 2.5 mM each dNTP (also see appendix 2D)
  • 5 U/µl Platinum Taq DNA polymerase (Invitrogen)
  • Mixture of 0.2 µM forward and reverse PCR primers (may be synthesized by Integrated DNA Technologies)
  • AcycloPrime FP SNP Detection Kit (Perkin‐Elmer) containing:
    • Exo‐SAP‐IT kit
    • AcycloPol DNA polymerase
    • AcycloTerminator mix (in combinations of two terminators labeled with R110 or TAMRA; the six combinations each contain two labeled terminators and the other two unlabeled terminators: R110‐G/TAMRA‐A; R110‐G/TAMRA‐C; R110‐G/TAMRA‐T; R110‐C/TAMRA‐A; R110‐C/TAMRA‐T; R110‐A/TAMRA‐T)
    • 10× reaction buffer
  • 1 µM SNP primer (see recipe)
  • 384‐well black PCR plates
  • Multichannel pipettor or liquid‐handling workstation
  • Sealing mats for 384‐well PCR plates
  • Thermal cycler with heated cover
  • FP plate reader: Victor2 or EnVision (Perkin‐Elmer); LJL Analyst (Molecular Devices); or Ultra (Tecan)
  • Software to create an xy scatter plot: e.g., Microsoft Excel or SNPScorer (Perkin‐Elmer)

Basic Protocol 2: Primer Extension Assay with 4‐Plex PCR

  Materials
  • 0.8 ng/µl genomic DNA of interest
  • 10× PCR buffer: 200 mM Tris·Cl, pH 8.4 ( appendix 2D)/500 mM KCl (may be purchased from Invitrogen)
  • 2.5 mM 4dNTP mix: 2.5 mM each dNTP (also see appendix 2D)
  • 5 U/µl Platinum Taq DNA polymerase (Invitrogen)
  • Mixture of 0.22 µM each of the eight PCR primers for the four SNPs in the set (may be synthesized by Integrated DNA Technologies)
  • AcycloPrime FP SNP Detection Kit (Perkin‐Elmer) containing:
    • Exo‐SAP‐IT kit
    • AcycloPol DNA polymerase
    • AcycloTerminator mix (in combinations of two terminators labeled with R110 or TAMRA; the six combinations each contain two labeled terminators and the other two unlabeled terminators: R110‐G/TAMRA‐A; R110‐G/TAMRA‐C; R110‐G/TAMRA‐T; R110‐C/TAMRA‐A; R110‐C/TAMRA‐T; R110‐A/TAMRA‐T)
    • 10× reaction buffer
  • 0.2 µM SNP primer (see recipe)
  • 384‐well clear PCR plates and black PCR plates
  • Multichannel pipettor or liquid handling workstation
  • Sealing mats for 384‐well PCR plates
  • Thermal cycler with heated cover
  • FP plate reader: Victor2 or EnVision (Perkin‐Elmer); Analyst (Molecular Devices); or Ultra (Tecan)
  • Software to create an xy scatter plot: e.g., Microsoft Excel or SNPScorer (Perkin‐Elmer)
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Figures

Videos

Literature Cited

Literature Cited
   Akula, N., Chen, Y.S., Hennessy, K., Schulze, T.G., Singh, G., and McMahon, F.J. 2002. Utility and accuracy of template‐directed dye‐terminator incorporation with fluorescence‐polarization detection for genotyping single nucleotide polymorphisms. BioTechniques 32:1072‐1078.
   Chen, X., Levine, L., and Kwok, P.‐Y. 1999. Fluorescence polarization in homogeneous nucleic acid analysis. Genome Res. 9:492‐498.
   Hsu, T.M. and Kwok, P.‐Y. 2003. Homogeneous primer extension assay with fluorescence polarization detection. Methods Mol. Biol. 212:177‐187.
   Hsu, T.M., Chen, X., Duan, S., Miller, R., and Kwok, P.‐Y. 2001a. A universal SNP genotyping assay with fluorescence polarization detection. BioTechniques 31:560‐570.
   Hsu, T.M., Law, S.M., Duan, S., Neri, B.P., and Kwok, P.‐Y. 2001b. Genotyping single nucleotide polymorphisms by the Invader assay with dual‐color fluorescence polarization detection. Clin. Chem. 47:1373‐1377.
   Kwok, P.‐Y. 2002. SNP genotyping with fluorescence polarization detection. Hum. Mutat. 19:315‐323.
   Latif, S., Bauer‐Sardia, I., Ranade, K., Livak, K., and Kwok, P.‐Y. 2001. Fluorescence polarization in homogeneous nucleic acid analysis. II. 5′‐nuclease assay. Genome Res. 11:436‐440.
   Nikiforov, T.T., Rendle, R.B., Goelet, P., Rogers, Y.H., Kotewicz, M.L., Anderson, S., Trainor, G.L., and Knapp, M.R. 1994. Genetic bit analysis: A solid phase method for typing single nucleotide polymorphisms. Nucl. Acids Res. 22:4167‐4175.
   Pastinen, T., Kurg, A., Metspalu, A., Peltonen, L., and Syvanen, A.C. 1997. Minisequencing: A specific tool for DNA analysis and diagnostics on oligonucleotide arrays. Genome Res. 7:606‐614.
   Perrin, F. 1926. Polarization de la lumière de fluorescence. Vie moyenne de molécules dans l'etat excité. J. Phys. Radium 7:390‐401.
   Syvanen, A.C. 1994. Detection of point mutations in human genes by the solid‐phase minisequencing method. Clin. Chim. Acta. 226:225‐236.
   Syvanen, A.C. 2001. Accessing genetic variation: Genotyping single nucleotide polymorphisms. Nat. Rev. Genet. 2:930‐942.
   Syvanen, A.C., Aalto‐Setala, K., Harju, L., Kontula, K., and Soderlund, H. 1990. A primer‐guided nucleotide incorporation assay in the genotyping of apolipoprotein E. Genomics 8:684‐692.
   Vieux, E.F., Kwok, P.‐Y., and Miller, R.D. 2002. Primer design for PCR and sequencing in high throughput analysis of SNPs. BioTechniques Jun. Suppl.:28‐32.
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