Random shearing as an alternative to digestion for mitochondrial DNA processing in droplet digital PCR.

Authors:
Mr Miguel Ramirez-Gaona, Bsc
Mr Miguel Ramirez-Gaona, Bsc
University of Alberta
Bioinformatics/Molecular Genetics
Edmonton, Alberta | Canada

Mitochondrion 2017 Jan 10;32:16-18. Epub 2016 Nov 10.

University of California San Diego, 9500 Gilman Drive, Stein Research Building Room 324, La Jolla, CA 92093, USA. Electronic address:

Droplet digital PCR (ddPCR) is a quantitative assay that requires DNA fragmentation to maximize reaction efficiency. Here, we measured the proportion of mitochondrial DNA (mtDNA) carrying the "common deletion," a rare event, to compare quantification sensitivities between alternative DNA fragmentation methods (sonication and QIAshredder spin columns) against enzymatic digestion (traditionally used). QIAshredder showed the highest sensitivity when compared to sonication, followed by digestion. Also, both sonication and QIAshredder fragmentation had shorter processing times than enzymatic digestion; therefore, QIAshredder fragmentation and sonication are alternative DNA processing methods that maximize ddPCR quantification for the detection of rare events.

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Source
http://dx.doi.org/10.1016/j.mito.2016.11.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5218872PMC
January 2017
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