Genotyping Using the TaqMan Assay

Lester Hui1, Terrye DelMonte1, Koustubh Ranade1

1 Bristol‐Myers Squibb, Princeton, New Jersey
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 2.10
DOI:  10.1002/0471142905.hg0210s56
Online Posting Date:  January, 2008
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Abstract

The 5′‐nuclease allelic discrimination assay, or TaqMan assay, is a PCR‐based assay for genotyping single nucleotide polymorphisms (SNPs). The region flanking the SNP is amplified in the presence of two allele‐specific fluorescent probes. The probes do not fluoresce in solution because of a quencher at the 3′ end. The presence of two probes allows the detection of both alleles in a single tube. Moreover, because probes are included in the PCR, genotypes are determined without any post‐PCR processing, a feature that is unavailable with most other genotyping methods. This unit describes probe and primer design and PCR conditions. Curr. Protoc. Hum. Genet. 56:2.10.1‐2.10.8. © 2008 by John Wiley & Sons, Inc.

Keywords: genotyping; TaqMan; SNP; AutoProbeMaker; PCR; fluorescent

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Genotyping Using the TaqMan Assay
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Genotyping Using the TaqMan Assay

  Materials
  • 15 ng/µl genomic DNA (sample and reference) in TE buffer, pH 8.0 ( appendix 2D)
  • PCR mix (see Table 2.10.2)
    Table 2.0.2   MaterialsPCR Mix for TaqMan Genotyping Using 96‐ or 384‐Well Format

    Stock solution a For one 96‐well reaction (µl) For one 384‐well reaction (µl)
    Forward primer 0.1125 0.048
    Reverse primer 0.1125 0.048
    FAM probe 0.0312 0.016
    VIC probe 0.0312 0.016
    2× master mix b 12.5 4.0
    Water 10.2 0.872
    Total per well 23 5
    For one 96‐well plate (µl) c For one 384‐well plate (µl) c
    Forward primer 13.5 26.4
    Reverse primer 13.5 26.4
    FAM probe 3.74 8.80
    VIC probe 3.74 8.80
    2× master mix b 1500 2200
    Water 1224 479.60
    Total per well 23 5

     aAll primers and probes are at a starting concentration of 100 µm.
     bThe 2× TaqMan master mix can be purchased from Applied Biosystems. A single lot of master mix should be used, since lot‐to‐lot variability can contribute to variation in fluorescence values.
     cCalculations based on making excess cocktail to allow for dead volume and pipetting errors.
  • 96‐well or 384‐well optical plates and plate sealers (Applied Biosystems)
  • Plate centrifuge
  • ABI PRISM 7700 sequence detection system for 96‐well format or ABI PRISM 7900 HT sequence detection system for 384‐well format (Applied Biosystems)
  • Thermal cycler 9700 (Applied Biosystems)
  • Plate stacker and robotic arm connected to ABI 7900 HT system (optional)
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Figures

Videos

Literature Cited

   Collins, F.S., Guyer, M.S., and Chakravarti, A. 1997. Variations on a theme: Cataloguing human DNA sequence variation. Science 278:1580‐1581.
   Kutyavin, I.V., Afonina, I.A., Mills, A., Gorn, V.V., Lukhtanov, E.A., Belousov, E.S., Singer, M.J., Walburger, D.K., Lokhov, S.G., Gall, A.A., Dempcy, R., Reed, M.W., Meyer, R.B., and Hedgpeth, J. 2000. 3′‐minor groove binder‐DNA probes increase sequence specificity at PCR extension temperatures. Nucl. Acids Res. 28:655‐661.
   Landegren, U., Nilsson, M., and Kwok, P.Y. 1998. Reading bits of genetics information: Methods for single‐nucleotide polymorphism analysis. Genome Res. 8:769‐776.
   Livak, K.J., Marmaro J., and Todd J.A. 1995. Towards fully automated genome‐wide polymorphism screening. Nat. Genet. 9:341‐342.
   Ranade, K., Chang, M.‐S., Ting, C.‐T., Pei, D., Hsiao, C.‐F., Olivier, M., Pesich, R., Hebert, J., Chen, Y.‐I., Dzau, V.J., Curb, D., Olshen, R., Risch, N., Cox, D.R., and Botstein, D. 2001. High‐throughput genotyping with single nucleotide polymorphisms. Genome Res. 11:1262‐1268.
   Risch, N. and Merikangas, K. 1996. The future of genetic studies of complex human diseases. Science 13:1516‐1517.
   Rozen, S. and Skaletsky, H. 2000. Primer3 on the WWW for general users and for biologist programmers. In Bioinformatics Methods and Protocols: Methods in Molecular Biology (S. Krawetz and S. Misener, eds.) pp 365‐386. Humana Press, Totowa, N.J.
Internet Resources
   http://www.seesnps.com/currentprotocols
  Free program to batch design Taqman genotyping assays.
   http://frodo.wi.mit.edu/primer3/input.htm
  Widely used open source program for designing PCR primers.
   http://www.appliedbiosystems.com
  Predeveloped assays, custom designed assays, and assay design software (Primer Express).
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