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

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Microchip Diagnostics

Part of the book series: Methods in Molecular Biology ((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.

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References

  1. Vogelstein B, Kinzler KW (2004) Cancer genes and the pathways they control. Nat Med 10:789–799

    Article  CAS  Google Scholar 

  2. Stratton MR, Campbell PJ et al (2009) The cancer genome. Nature 458(7239):719–724

    Article  CAS  Google Scholar 

  3. Diehl F, Diaz LA (2007) Digital quantification of mutant DNA in cancer patients. Curr Opin Oncol 19(1):36–42

    Article  CAS  Google Scholar 

  4. Sawyers CL (2008) Cancer biomarker problem. Nature 452:548–552

    Article  CAS  Google Scholar 

  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):542–548

    Article  CAS  Google Scholar 

  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):579–584

    Article  CAS  Google Scholar 

  7. Milbury CA, Li J, Makrigiorgos MG (2009) PCR-based methods for the enrichment of minority alleles and mutations. Clin Chem 55(4):632–640

    Article  CAS  Google Scholar 

  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):180–186

    Article  Google Scholar 

  9. Foy CA, Parkes HC (2001) Emerging homogeneous DNA-based technologies in the clinical laboratory. Clin Chem 47(6):990–1000

    CAS  Google Scholar 

  10. Whitcombe D, Newton CR, Little S (1998) Advances in approaches to DNA-based diagnostics. Curr Opin Biotechnol 9(6):602–608

    Article  CAS  Google Scholar 

  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):1169–1179

    Article  CAS  Google Scholar 

  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):166–168

    Article  CAS  Google Scholar 

  13. Ogino S, Kawasaki T, Brahmandam M et al (2005) Sensitive sequencing method for KRAS mutation detection by pyrosequencing. J Mol Diagn 7(3):413–421

    Article  CAS  Google Scholar 

  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):374–379

    Article  CAS  Google Scholar 

  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):292–299

    Article  CAS  Google Scholar 

  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):425–432

    Article  CAS  Google Scholar 

  17. Sykes PJ, Neoh SH, Brisco MJ et al (1992) Quantitation of targets for PCR by use of limiting dilution. Biotechniques 13(3):444–449

    CAS  Google Scholar 

  18. Vogelstein B, Kinzler KW (1999) Digital PCR. Proc Natl Acad Sci U S A 96(16):9236–9241

    Article  CAS  Google Scholar 

  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):489–498

    Article  CAS  Google Scholar 

  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):858–886

    Article  CAS  Google Scholar 

  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):884–890

    Article  CAS  Google Scholar 

  22. Shendure J, Porreca GJ, Reppas NB et al (2005) Accurate multiplex polony sequencing of an evolved bacterial genome. Science 309:1728–1732

    Article  CAS  Google Scholar 

  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 24:3769–3777

    Article  CAS  Google Scholar 

  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 100:8817–8822

    Article  CAS  Google Scholar 

  25. Li M, Diehl F, Dressman D et al (2006) Beaming up for detection and quantification of rare sequence variants. Nat Methods 3(2):95–97

    Article  CAS  Google Scholar 

  26. Nakano M, Stone HA, Komatsu GPJ et al (2005) Single-molecule PCR using water-in-oil emulsion. J Biotechnol 102:117–124

    Article  Google Scholar 

  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:e109

    Article  Google Scholar 

  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):2156–2166

    Article  CAS  Google Scholar 

  29. Perkins G, Lu H, Garlan F, Taly V (2017) Droplet-Based Digital PCR: Application in Cancer Research. Adances in Clinical Chemistry (85): 44–91

    Google Scholar 

  30. Holtze C, Rowat AC, Agresti JJ et al (2008) Biocompatible surfactants for water-in- fluorocarbon emulsions. Lab Chip 8(10):1632–1639

    Article  CAS  Google Scholar 

  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):3992–3995

    Article  CAS  Google Scholar 

  32. Xia YN, Whitesides GM (2006) Soft lithography. Angew. Chem. Int. Ed 37(5):551–575

    Google Scholar 

  33. Anna SL, Bontoux N, Stone HA (2003) Formation of dispersions using flow focusing in microchannels. Appl Phys Lett 82:364–366

    Article  CAS  Google Scholar 

  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: 2902–2908

    Google Scholar 

  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):e7464

    Article  Google Scholar 

  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):4813–4821

    Article  CAS  Google Scholar 

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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).

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Correspondence to Valerie Taly .

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Pekin, D., Taly, V. (2017). Droplet-Based Microfluidics Digital PCR for the Detection of KRAS Mutations. In: Taly, V., Viovy, JL., Descroix, S. (eds) Microchip Diagnostics. Methods in Molecular Biology, vol 1547. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6734-6_12

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  • DOI: https://doi.org/10.1007/978-1-4939-6734-6_12

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6732-2

  • Online ISBN: 978-1-4939-6734-6

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