title: Random shearing as an alternative to digestion for mitochondrial DNA processing in droplet digital PCR.
abstract: 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.
authors.affiliation: University of California San Diego, 9500 Gilman Drive, Stein Research Building Room 324, La Jolla, CA 92093, USA. Electronic address: avitomirov@ucsd.edu.
authors.forename: Andrej
authors.initials: A
authors.lastname: Vitomirov
authors.affiliation: University of Alberta, 116 Street & 85 Avenue, Edmonton, AB T6G 2R3, Canada. Electronic address: miguel1@ualberta.ca.
authors.forename: Miguel
authors.initials: M
authors.lastname: Ramirez-Gaona
authors.affiliation: University of California San Diego, 9500 Gilman Drive, Stein Research Building Room 324, La Jolla, CA 92093, USA; Veterans Administration San Diego Healthcare System, 3350 La Jolla Village Drive, San Diego, CA 92161, USA. Electronic address: srmehta@ucsd.edu.
authors.forename: Sanjay R
authors.initials: SR
authors.lastname: Mehta
authors.affiliation: University of California San Diego, 9500 Gilman Drive, Stein Research Building Room 324, La Jolla, CA 92093, USA. Electronic address: joperez@ucsd.edu.
authors.forename: Josué
authors.initials: J
authors.lastname: Pérez-Santiago
mesh_terms: D004272:DNA, Mitochondrial / Q000235:genetics*; D006868:Hydrolysis; D028361:Mitochondrial Diseases / Q000175:diagnosis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013010:Sonication; D013048:Specimen Handling / Q000379:methods*; D013997:Time Factors
chemical_list: D004272:DNA, Mitochondrial
keywords: Droplet digital PCR; Mitochondrial DNA; Mitochondrial common deletion; QIAshredder; Sonication