Advertisement

Digital PCR and the QuantStudio™ 3D Digital PCR System

  • Marion LaigEmail author
  • Christie Fekete
  • Nivedita Majumdar
Protocol
  • 1.3k Downloads
Part of the Methods in Molecular Biology book series (MIMB, volume 2065)

Abstract

The great promise of digital PCR is the potential for unparalleled precision enabling accurate measurements for detection and quantification of genetic material. This chapter walks the reader through the fundamentals of digital PCR technology including digital PCR modeling using Poisson statistics. It describes a highly successful implementation of digital PCR technology using the chip-based nanofluidic Applied Biosystems™ QuantStudio™ 3D digital PCR system. It reviews the large number of applications where digital PCR is poised to make significant impacts. These include applications where detection of rare genetic targets is prioritized such as liquid biopsy, rare mutation detection, confirmation of NGS variant detection, detection of fusion transcripts, detection of chimerism and GMO detection and monitoring. These further include applications where accurate quantification of genetic targets is prioritized such as generation of references and standards, copy number variation, and NGS Library quantification.

Key words

Digital PCR Poisson Limit of detection Rare mutation Copy number GMO 

Notes

Acknowledgments

For Research Use Only. Not for use in diagnostic procedures. We thank David Keys and Ross Stolsmark for thoughtful review of the manuscript.

References

  1. 1.
    Vogelstein B, Kinzler KW (1999) Digital PCR. Proc Natl Acad Sci U S A 96:9236–9241CrossRefGoogle Scholar
  2. 2.
    Simmonds P, Balfe P, Peutherer JF et al (1990) Human immunodeficiency virus-infected individuals contain provirus in small numbers of peripheral mononuclear cells and at low copy numbers. J Virol 64:864–872PubMedPubMedCentralGoogle Scholar
  3. 3.
    Majumdar N, Wessel T, Marks J (2015) Digital PCR modeling for maximal sensitivity, dynamic range and measurement precision. PLoS One 10:1–17CrossRefGoogle Scholar
  4. 4.
    Majumdar N, Banerjee S, Pallas M et al (2017) Poisson plus quantification for digital PCR systems. Sci Rep 7:2045–2322CrossRefGoogle Scholar
  5. 5.
    Dietrich D, Uhl B, Sailer V et al (2013) Improved PCR performance using template DNA from formalin-fixed and paraffin-embedded tissues by overcoming PCR inhibition. PLoS One 8(10):e77771. eCollection 2013CrossRefGoogle Scholar
  6. 6.
    Publication COL04004 0417 (2017) Rare mutation analysis using the QuantStudio 3D Digital PCR system. Quick Reference Protocol. Thermo Fisher ScientificGoogle Scholar
  7. 7.
    Huang FW, Hodis E, Xu MJ et al (2013) Highly recurrent TERT promoter mutations in human melanoma. Science 339:957–959CrossRefGoogle Scholar
  8. 8.
    Bardelli A, Pantel K (2017) Liquid biopsies, what we do not know (yet). Cancer Cell 31:172–179CrossRefGoogle Scholar
  9. 9.
    Crowley E, Di Nicolantonio F, Loupakis F et al (2013) Liquid biopsy: monitoring cancer-genetics in the blood. Nat Rev Clin Oncol 10:472–484CrossRefGoogle Scholar
  10. 10.
    Garcia-Saenz JA, Ayllon P, Laig M et al (2017) Tumor burden monitoring using cell-free tumor DNA could be limited by tumor heterogeneity in advanced breast cancer and should be evaluated together with radiographic imaging. BMC Cancer 17:210CrossRefGoogle Scholar
  11. 11.
    Yates LR, Campbell PJ (2012) Evolution of the cancer genome. Nat Rev Genet 13:795–806CrossRefGoogle Scholar
  12. 12.
    Roedel A, Laig M, Schmitz A (2016) Detection of the TMPRSS2:ERG fusion transcript. Application Note, Thermo Fisher Scientific, Publication COL31191 0616Google Scholar
  13. 13.
    Tomlins SA, Laxman B, Varambally S et al (2008) Role of the TMPRSS2-ERG gene fusion in prostate cancer. Neoplasia 10:177–188CrossRefGoogle Scholar
  14. 14.
    Friedman D (2013) Are engineered foods evil? Sci Am 309:80–85CrossRefGoogle Scholar
  15. 15.
    Gerry C (2015) Feeding the world one genetically modified tomato at a time: a scientific perspective. Blog, special edition on GMOs. http://sitn.hms.harvard.edu/flash/2015/feeding-the-world/)
  16. 16.
    Wan JR, Song L, Wu YL et al (2016) Application of digital PCR in the analysis of transgenic soybean plants. Adv Biosci Biotechnol 7:403–417CrossRefGoogle Scholar
  17. 17.
    Almal SH, Padh H (2012) Implications of gene copy number variation in health and diseases. J Hum Genet 57:6–13CrossRefGoogle Scholar
  18. 18.
    Inaki K, Liu ET (2012) Structural mutations in cancer: mechanistic and functional insights. Trends Genet 28:550–559CrossRefGoogle Scholar
  19. 19.
    Publication CO09771 0514 (2014) Copy number variation in breast cancer translational research. Publication CO09771 0514, Thermofisher.com
  20. 20.
    Publication CO020253 0216 (2016). Precise quantification of Ion Torrent libraries on the QuantStudio 3D Digital PCR system. Application Note, Thermofisher.com
  21. 21.
    Publication CO35377 1014 (2014). Precise quantification of Illumina® libraries on the QuantStudio 3D Digital PCR System. Application Note, Thermofisher.com
  22. 22.
    Publication CO020251 0216 (2016) Copy number variation analysis using the QuantStudio 3D Digital PCR System. Thermofisher.com

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Marion Laig
    • 1
    Email author
  • Christie Fekete
    • 1
  • Nivedita Majumdar
    • 1
  1. 1.Thermo Fisher ScientificSouth San FranciscoUSA

Personalised recommendations