Developing Integrated Approaches To Mitochondrial DNA Analysis Using Microfluidic Chips

• Author / Creator

  Northrup, Victoria A.

• Mutations in the mitochondrial genome (mtDNA) have been linked to a wide variety of disorders. mtDNA analyses require large starting samples and are complicated by the presence of nuclear mitochondrial pseudogenes (NUMTs), which can cause false positives in PCR-based approaches. Microfluidic chips (MFCs) allow assays, which are typically run at the laboratory bench, to be performed on small microchips, thus facilitating high-throughput analyses. We have used MFCs to analyze plasmid DNA (pDNA) and mtDNA, in an attempt to develop mtDNA diagnostic capabilities using small sample volumes.
  
  We were able to successfully manipulate 1 pg of pDNA, the approximate amount of mtDNA in 1500 fibroblasts, which is the lowest concentration of DNA to be successfully manipulated on MFCs that we are aware of. Based on this plasmid isolation we were also able to develop a MFC-based plasmid miniprep using 5 orders of magnitude fewer starting cells. Modification of these approaches resulted in the isolation of mtDNA from fibroblasts and leukocytes, albeit with some nuclear DNA (nDNA) contamination.
  
  Using the Supercoiled DNA (SC) ladder, we were able to develop a capillary electrophoresis (CE)-based plasmid separation that could resolve two plasmids with a 1.5 kb size difference. This resolution is sufficient to allow identification of the vast majority of reported mtDNA deletions. These experiments reported here therefore lay the groundwork for performing mtDNA diagnostics on MFCs.

• Subjects / Keywords

  • Microfluidic Chips
  • Plasmids
  • Mitochondrial DNA
  • Biotechnology
  • Capillary Electrophoresis
  • Lab on a chip
  • Plasmid miniprep

• Graduation date

  Spring 2015

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R3534Z

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.