Bi-Allelic SNP Genotyping Using the TaqMan® Assay

  • John Woodward
Part of the Methods in Molecular Biology book series (MIMB, volume 1145)


With TaqMan® technology allele-specific probes are utilized for quick and reliable genotyping of known polymorphic sites. TaqMan assays are robust in genotyping multiple variant types, including single nucleotide polymorphisms, insertions/deletions, and presence/absence variants. To query a single bi-allelic polymorphism, two TaqMan probes labeled with distinct fluorophores are designed such that they hybridize to different alleles during PCR-based amplification of a surrounding target region. During the primer extension phase of PCR, the 5′–3′ exonuclease activity of Taq polymerase cleaves and releases the fluorophores from bound probes. At the end of PCR, the emission intensity of each fluorophore is measured and allele determination at the queried site can be made.

Key words

5′ Nuclease assay Applied biosystems 6-FAMTM VIC® Single nucleotide polymorphism Genotyping 


  1. 1.
    Kaeppler S (2012) Heterosis: many genes, many mechanisms—end the search for an undiscovered unifying theory. ISRN Botany, 2012. doi: 10.5402/2012/682824
  2. 2.
    Tester M, Langridge P (2010) Breeding technologies to increase crop production in a changing world. Science 327:818–822PubMedCrossRefGoogle Scholar
  3. 3.
    Eathington SR, Crosbie TM, Edwards MD et al (2007) Molecular markers in a commercial breeding program. Crop Sci 47(3):154Google Scholar
  4. 4.
    Moose SP, Mumm RH (2008) Molecular plant breeding as the foundation for 21st century crop improvement. Plant Physiol 147:969–977PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Rafalski A (2002) Applications of single nucleotide polymorphisms in crop genetics. Curr Opin Plant Biol 5:94–100PubMedCrossRefGoogle Scholar
  6. 6.
    Holland PM, Abramson RD, Watson R et al (1991) Detection of specific polymerase chain reaction product by utilizing the 5′–3′ exonuclease activity of Thermus aquaticus DNA polymerase. PNAS 88:7276–7280PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Livak KJ (1999) Allelic discrimination using fluorogenic probes and the 5′ nuclease assay. Genet Anal 14:143–149PubMedCrossRefGoogle Scholar
  8. 8.
    Kutyavin IV, Afonina IA, Mills A et al (2000) 3′-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures. Nucleic Acids Res 28:655–661PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–386PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.DuPont PioneerJohnstonUSA

Personalised recommendations