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

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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.

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Acknowledgements

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

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Authors and Affiliations

  1. Department of Mechanical Engineering, Johns Hopkins University, Baltimore, MD, 21218, USA

    Alejandro Stark, Dong Jin Shin & Tza-Huei Wang

  2. Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, 21218, USA

    Tza-Huei Wang

  3. Johns Hopkins Institute for NanoBioTechnology, Baltimore, MD, 21218, USA

    Tza-Huei Wang

Authors
  1. Alejandro Stark
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  2. Dong Jin Shin
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  3. Tza-Huei Wang
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Correspondence to Tza-Huei Wang.

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Stark, A., Shin, D.J. & Wang, TH. A sample-to-answer droplet magnetofluidic assay platform for quantitative methylation-specific PCR. Biomed Microdevices 20, 31 (2018). https://doi.org/10.1007/s10544-018-0276-6

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  • DOI: https://doi.org/10.1007/s10544-018-0276-6

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