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Permanent link (DOI): https://doi.org/10.7939/R3534Z

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Developing Integrated Approaches To Mitochondrial DNA Analysis Using Microfluidic Chips Open Access

Descriptions

Other title
Subject/Keyword
Biotechnology
Microfluidic Chips
Capillary Electrophoresis
Plasmid miniprep
Mitochondrial DNA
Lab on a chip
Plasmid
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Northrup, Victoria A.
Supervisor and department
Backhouse, Christopher (Electrical and Computer Engineering)
Rachubinski, Richard (Cell Biology)
Glerum, D. Moira (Cell Biology)
Examining committee member and department
Simmen, Thomas (Cell Biology)
Backhouse, Christopher (Electrical and Computer Engineering)
Glerum, D. Moira (Cell Biology)
Dacks, Joel (Cell Biology)
Hume, Stacey (Medical Genetics)
Rachubinski, Richard (Cell Biology)
Department
Department of Cell Biology
Specialization

Date accepted
2014-11-24T13:48:41Z
Graduation date
2015-06
Degree
Master of Science
Degree level
Master's
Abstract
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.
Language
English
DOI
doi:10.7939/R3534Z
Rights
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
Citation for previous publication
Northrup, V.A., C.J. Backhouse, and D.M. Glerum. 2010. Development of a microfluidic chip-based plasmid miniprep. Anal. Biochem. 402: 185-190.Ma, T.*, V. Northrup*, A.O. Fung, D.M. Glerum, and C.J. Backhouse. 2012. Polymeric rapid prototyping for inexpensive and portable medical diagnostics. In Photonics North 2012. International Society for Optics and Photonics. 84140B-84120B-84128. *These authors contributed equally.

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