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Nucleic Acid Amplification for Accessible Diagnostics of Chronic and Acute Conditions

  • Author(s): Hambalek, Jacob Amos
  • Advisor(s): Di Carlo, Dino
  • et al.
Abstract

Nucleic acid amplification is an established strategy for detecting trace amounts of genetic material. Assays employing nucleic acid amplification are prevalent throughout several fields of life science, particularly for diagnostic applications. Advances in access to nucleic acid amplification are necessary to increase its prevalence in laboratory settings and further enable its applicability for point-of-care testing. The work described herein demonstrates several advancements in increasing access to nucleic acid amplification testing. One advancement we demonstrate is an approach for rapid and portable assessment of DNA base methylation, an emerging biomarker correlated to chronic conditions such as cardiovascular disease. This approach incorporates methylation sensitive restriction endonuclease digestion with an isothermal mechanism for nucleic acid amplification to determine the presence of methylation marks at DNA targets of interest. We additionally demonstrate a platform for digital nucleic acid amplification in microparticle templated water-in-oil emulsions. Utilizing microparticles with cavitary geometries for improved loading of DNA templates, this digital nucleic acid amplification approach requires no specialized equipment and indicates improved reaction efficiency over comparable microparticle platforms. We envision the coalescence of these and other nucleic acid amplification advancements for the democratization of amplification-based technologies to improve the capability and accessibility of nucleic acid diagnostic testing.

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