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A sample-to-answer droplet magnetofluidic assay platform for quantitative methylation-specific PCR

  • Alejandro Stark
  • Dong Jin Shin
  • Tza-Huei Wang
Article

Abstract

Dysregulation of DNA methylation has been identified as an epigenetic biomarker for numerous cancer types. Gene-specific identification techniques relying on methylation-specific PCR (MSP) require a lengthy manual benchtop process that is susceptible to human-error and contamination. This MSP assay requires a series of discrete sample processing steps including genomic DNA extraction, bisulfite conversion and readout via PCR. In this work, we present a streamlined assay platform utilizing droplet magnetofluidic principles for integration of all sample processing steps required to obtain quantitative MSP signal from raw biological samples. We present a streamlined protocol for solid-phase extraction and bisulfite conversion of genomic DNA, which minimizes reagent use and simplifies the sample preparation protocol for implementation on a compact assay platform. Furthermore, we present a thermally robust assay chip that enables DNA extraction, bisulfite conversion and quantitative PCR from biological samples on a single device. Technical improvements to facilitate DNA extraction and PCR on a single chip in addition to chip performance characterization data are presented.

Keywords

Methylation Epigenetics Methylation-specific PCR Lab-on-chip Magnetofluidics qPCR Biomarkers 

Notes

Acknowledgements

The authors would also like to thank funding sources from National Institutes of Health (R21CA186809, UG3CA211457, U01CA200499).

References

  1. V.J. Bailey, Y. Zhang, B.P. Keeley, C. Yin, K.L. Pelosky, M.V. Brock, S.B. Baylin, J.G. Herman, T.-H. Wang, Clin. Chem. 56, 1022 (2010)CrossRefGoogle Scholar
  2. S.B. Baylin, J.G. Herman, Trends Genet. 16, 168 (2000)CrossRefGoogle Scholar
  3. A.P. Bird, Nature 321, 209 (1985)CrossRefGoogle Scholar
  4. P.M. Das, R. Singal, J. Clin. Oncol. 22, 4632 (2004)CrossRefGoogle Scholar
  5. S. Derks, M.H.F.M. Lentjes, D.M.E.I. Hellebrekers, A.P. de Bruïne, J.G. Herman, M. van Engeland, Cell. Oncol. 26, 291 (2004)Google Scholar
  6. H. Donninger, M.D. Vos, G.J. Clark, J. Cell Sci. 120, 3163 (2007)CrossRefGoogle Scholar
  7. M. Ehrlich, Oncogene 21, 5400 (2002)CrossRefGoogle Scholar
  8. M.F. Fraga, M. Esteller, BioTechniques 33, 632 (2002)Google Scholar
  9. M. Frommer, L.E. McDonald, D.S. Millar, C.M. Collis, F. Watt, G.W. Grigg, P.L. Molloy, C.L. Paul, Proc. Natl. Acad. Sci. U. S. A. 89, 1827 (1992)CrossRefGoogle Scholar
  10. J.G. Herman, S.B. Baylin, N. Engl. J. Med. 349, 2042 (2003)CrossRefGoogle Scholar
  11. J.G. Herman, F. Latif, Y. Weng, M.I. Lerman, B. Zbar, S. Liu, D. Samid, D.S. Duan, J.R. Gnarra, W.M. Linehan, Proc. Natl. Acad. Sci. U. S. A. 91, 9700 (1994)CrossRefGoogle Scholar
  12. J.G. Herman, J.R. Graff, S. Myohanen, B.D. Nelkin, S.B. Baylin, S. Myöhänen, B.D. Nelkin, S.B. Baylin, Proc. Natl. Acad. Sci. U. S. A. 93, 9821 (1996)CrossRefGoogle Scholar
  13. S. Kalari and G. P. Pfeifer, Identification of Driver and Passenger DNA Methylation in Cancer by Epigenomic Analysis (2010)Google Scholar
  14. B.P. Keeley, A. Stark, T.R. Pisanic, R. Kwak, Y. Zhang, J. Wrangle, S.B. Baylin, J.G. Herman, N. Ahuja, M.V. Brock, T.-H.H. Wang, T.R. Pisanic II, R. Kwak, Y. Zhang, J. Wrangle, S.B. Baylin, J.G. Herman, N. Ahuja, M.V. Brock, T.-H.H. Wang, Clin. Chim. Acta 425, 169 (2013)CrossRefGoogle Scholar
  15. A. Merlo, J.G. Herman, L. Mao, D.J. Lee, E. Gabrielson, P.C. Burger, S.B. Baylin, D. Sidransky, Nat. Med. 1, 686 (1995)CrossRefGoogle Scholar
  16. S. Park, Y. Zhang, T.-H. Wang, S. Yang, Lab Chip 11, 2893 (2011)CrossRefGoogle Scholar
  17. N. Ramalingam, H.-B. Liu, C.-C. Dai, Y. Jiang, H. Wang, Q. Wang, K.M. Hui, H.-Q. Gong, Biomed. Microdevices 11, 1007 (2009)CrossRefGoogle Scholar
  18. D.J. Shin, P. Athamanolap, L. Chen, J. Hardick, M. Lewis, Y.H. Hsieh, R.E. Rothman, C.A. Gaydos, T.H. Wang, Sci. Rep. 7, 4495 (2017)CrossRefGoogle Scholar
  19. A. Stark, D.J. Shin, T.R. Pisanic II, K. Hsieh, T.-H. Wang, Biomed. Microdevices 18, 1 (2016)CrossRefGoogle Scholar
  20. Y. Zhang, T.-H. Wang, Adv. Mater. 25, 2903 (2013)CrossRefGoogle Scholar
  21. Y. Zhang, S. Park, S. Yang, T.-H.H. Wang, Biomed. Microdevices 12, 1043 (2010)CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Alejandro Stark
    • 1
  • Dong Jin Shin
    • 1
  • Tza-Huei Wang
    • 1
    • 2
    • 3
  1. 1.Department of Mechanical EngineeringJohns Hopkins UniversityBaltimoreUSA
  2. 2.Department of Biomedical EngineeringJohns Hopkins UniversityBaltimoreUSA
  3. 3.Johns Hopkins Institute for NanoBioTechnologyBaltimoreUSA

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