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

[摘要] 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.

[发布机构] University of Alberta

[效力级别] Microfluidic Chips