Advertisement

Droplet-Based Microfluidics Digital PCR for the Detection of KRAS Mutations

  • Deniz Pekin
  • Valerie TalyEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1547)

Abstract

We demonstrate an accurate and sensitive quantification of mutated KRAS oncogene in genomic DNA, using droplet-based microfluidics and digital PCR.

Key words

Droplet microfluidics Digital PCR Biomarkers KRAS Cancer 

Notes

Acknowledgments

This work was supported by Région Alsace, the Ministère de l'Enseignement Supérieur et de la Recherche, the Université de Strasbourg, the Université Paris Descartes, the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé et de la Recherche Médicale (INSERM), the Institut National du Cancer (INCa, no. 2009-1-RT-03-US-1) and the Association pour la recherche sur le Cancer (ARC, no. SL220100601375).

References

  1. 1.
    Vogelstein B, Kinzler KW (2004) Cancer genes and the pathways they control. Nat Med 10:789–799CrossRefGoogle Scholar
  2. 2.
    Stratton MR, Campbell PJ et al (2009) The cancer genome. Nature 458(7239):719–724CrossRefGoogle Scholar
  3. 3.
    Diehl F, Diaz LA (2007) Digital quantification of mutant DNA in cancer patients. Curr Opin Oncol 19(1):36–42CrossRefGoogle Scholar
  4. 4.
    Sawyers CL (2008) Cancer biomarker problem. Nature 452:548–552CrossRefGoogle Scholar
  5. 5.
    Lecomte T, Berger A, Zinzindohou F et al (2002) Detection of free circulating tumor-associated DNA in plasma of colorectal cancer patients and its association with prognosis. Int J Cancer 100(5):542–548CrossRefGoogle Scholar
  6. 6.
    Li J, Wang L, Mamon H, Kulke MH et al (2008) Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing. Nat Med 14(5):579–584CrossRefGoogle Scholar
  7. 7.
    Milbury CA, Li J, Makrigiorgos MG (2009) PCR-based methods for the enrichment of minority alleles and mutations. Clin Chem 55(4):632–640CrossRefGoogle Scholar
  8. 8.
    Caen O, Nizard P et al (2015) Digital PCR compartmentalization II. Contribution for the quantitative detection of circulating tumor DNA. Med Sci (Paris) 31(2):180–186CrossRefGoogle Scholar
  9. 9.
    Foy CA, Parkes HC (2001) Emerging homogeneous DNA-based technologies in the clinical laboratory. Clin Chem 47(6):990–1000Google Scholar
  10. 10.
    Whitcombe D, Newton CR, Little S (1998) Advances in approaches to DNA-based diagnostics. Curr Opin Biotechnol 9(6):602–608CrossRefGoogle Scholar
  11. 11.
    Carotenuto P, Roma C, Rachiglio AM et al (2010) Detection of KRAS mutations in colorectal carcinoma patients with an integrated PCR/sequencing and real-time PCR approach. Pharmacogenomics 11(8):1169–1179CrossRefGoogle Scholar
  12. 12.
    Dufort S, Richard MJ, de Fraipont F (2009) Pyrosequencing method to detect KRAS mutation in formalin-fixed and paran-embedded tumor tissues. Anal Biochem 391(2):166–168CrossRefGoogle Scholar
  13. 13.
    Ogino S, Kawasaki T, Brahmandam M et al (2005) Sensitive sequencing method for KRAS mutation detection by pyrosequencing. J Mol Diagn 7(3):413–421CrossRefGoogle Scholar
  14. 14.
    Lievre A, Bachet JB, Valrie Boige V et al (2008) KRAS mutations as an independent prognostic factor in patients with advanced colorectal cancer treated with cetuximab. J Clin Oncol 26(3):374–379CrossRefGoogle Scholar
  15. 15.
    Angulo B, Garca-Garca E, Martinez R et al (2010) A commercial real-time PCR kit provides greater sensitivity than direct sequencing to detect KRAS mutations: a morphology-based approach in colorectal carcinoma. J Mol Diagn 12(3):292–299CrossRefGoogle Scholar
  16. 16.
    Tsiatis AC, Norris-Kirby A, Rich RG et al (2010) Comparison of sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS mutations: diagnostic and clinical implications. J Mol Diagn 12(4):425–432CrossRefGoogle Scholar
  17. 17.
    Sykes PJ, Neoh SH, Brisco MJ et al (1992) Quantitation of targets for PCR by use of limiting dilution. Biotechniques 13(3):444–449Google Scholar
  18. 18.
    Vogelstein B, Kinzler KW (1999) Digital PCR. Proc Natl Acad Sci U S A 96(16):9236–9241CrossRefGoogle Scholar
  19. 19.
    Diehl F, Schmidt K, Durkee KH et al (2008) Analysis of mutations in DNA isolated from plasma and stool of colorectal cancer patients. Gastroenterology 135(2):489–498CrossRefGoogle Scholar
  20. 20.
    Dong SM, Traverso G, Johnson C et al (2001) Detecting colorectal cancer in stool with the use of multiple genetic targets. J Natl Cancer Inst 93(11):858–886CrossRefGoogle Scholar
  21. 21.
    Sefrioui D, Sarafan-Vasseur N et al (2015) Clinical value of chip-based digital-PCR platform for the detection of circulating DNA in metastatic colorectal cancer. Dig Liver Dis 47(10):884–890CrossRefGoogle Scholar
  22. 22.
    Shendure J, Porreca GJ, Reppas NB et al (2005) Accurate multiplex polony sequencing of an evolved bacterial genome. Science 309:1728–1732CrossRefGoogle Scholar
  23. 23.
    Leamon JH, Lee WL, Tartaro KR et al (2003) A massively parallel picotiter plate based platform for discrete picoliter-scale polymerase chain reactions. Electrophoresis 24:3769–3777CrossRefGoogle Scholar
  24. 24.
    Dressman D, Yan H, Traverso G et al (2003) Transforming single DNA molecules into uorescent magnetic particles for detection and enumeration of genetic variations. Proc Natl Acad Sci U S A 100:8817–8822CrossRefGoogle Scholar
  25. 25.
    Li M, Diehl F, Dressman D et al (2006) Beaming up for detection and quantification of rare sequence variants. Nat Methods 3(2):95–97CrossRefGoogle Scholar
  26. 26.
    Nakano M, Stone HA, Komatsu GPJ et al (2005) Single-molecule PCR using water-in-oil emulsion. J Biotechnol 102:117–124CrossRefGoogle Scholar
  27. 27.
    Chen W, Balaj L et al (2013) BEAMing and droplet digital PCR analysis of mutant IDH1 mRNA in Glioma patient serum and cerebrospinal fluid extracellular vesicles. Mol Ther Nucleic Acids 2:e109CrossRefGoogle Scholar
  28. 28.
    Pekin D, Skhiri Y, Baret J-C et al (2011) Quantitative and sensitive detection of rare mutations using droplet-based microfluidics. Lab Chip 11(13):2156–2166CrossRefGoogle Scholar
  29. 29.
    Perkins G, Lu H, Garlan F, Taly V (2017) Droplet-Based Digital PCR: Application in Cancer Research. Adances in Clinical Chemistry (85): 44–91Google Scholar
  30. 30.
    Holtze C, Rowat AC, Agresti JJ et al (2008) Biocompatible surfactants for water-in- fluorocarbon emulsions. Lab Chip 8(10):1632–1639CrossRefGoogle Scholar
  31. 31.
    Lievre A, Bachet JB, Le Corre D et al (2006) KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer. Cancer Res 66(8):3992–3995CrossRefGoogle Scholar
  32. 32.
    Xia YN, Whitesides GM (2006) Soft lithography. Angew. Chem. Int. Ed 37(5):551–575Google Scholar
  33. 33.
    Anna SL, Bontoux N, Stone HA (2003) Formation of dispersions using flow focusing in microchannels. Appl Phys Lett 82:364–366CrossRefGoogle Scholar
  34. 34.
    Mazutis L, Baret J-C, Treacy P et al (2009) Multi-step microfluidic droplet processing: kinetic analysis of an in vitro translated enzyme. Lab Chip 9: 2902–2908Google Scholar
  35. 35.
    Soh J, Okumura N, Lockwood WW et al (2009) Oncogene mutations, copy number gains and mutant allele specific imbalance (MASI) frequently occur together in tumor cells. PLoS One 4(10):e7464CrossRefGoogle Scholar
  36. 36.
    Mazutis L, Fallah Araghi A, Miller OJ et al (2009) Droplet-based microfluidic systems for high-throughput single DNA molecule isothermal amplification and analysis. Anal Chem 81(12):4813–4821CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.CNRS, Univ. Bordeaux, CRPP, UPR 8641PessacFrance
  2. 2.INSERM UMRS1147, CNRS SNC 5014, Université Paris DescartesParisCedex 06France

Personalised recommendations