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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.

SUBMITTER: Vitomirov A

PROVIDER: S-EPMC5218872 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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

Vitomirov Andrej A Ramirez-Gaona Miguel M Mehta Sanjay R SR Pérez-Santiago Josué J

Mitochondrion 20161110

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. Al ...[more]

PMID: 27838478

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