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

Date Published:
2017 Jan

Publication Type:
Journal Article

Authors:

Secondary:
Mitochondrion

Volume:
32

Pagination:
16-18

PMID:
27838478

URL:
https://pubmed.ncbi.nlm.nih.gov/27838478

DOI:
10.1016/j.mito.2016.11.005

Keywords:
DNA, Mitochondrial;Hydrolysis;Mitochondrial Diseases;Polymerase Chain Reaction;Sensitivity and Specificity;Sonication;Specimen Handling;Time Factors

Abstract:
<p>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.</p>