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

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• 作者：Andrej Vitomirov^a ; ^{avitomirov@ucsd.edu} ; Miguel Ramirez-Gaona^b ; ^{miguel1@ualberta.ca} ; Sanjay R. Mehta^a ; ^c ; ¹ ; ^{srmehta@ucsd.edu} ; Josué ; Pé ; rez-Santiago^a ; ¹ ; ^{joperez@ucsd.edu}
• 关键词：DdPCR ; droplet digital PCR ; mtDNA ; mitochondrial DNA ; TE ; Tris-EDTA ; ND2 ; mitochondrial NADH  ; + dehydrogenase 2 gene ; COM-DEL ; mitochondrial DNA &ldquo ; common deletion&rdquo ; RPP30 ; ribonuclease P protein subunit p30 gene
• 刊名：Mitochondrion
• 出版年：2017
• 出版时间：January 2017
• 年：2017
• 卷：32
• 期：Complete
• 页码：16-18
• 全文大小：369 K
• 卷排序：32

文摘

Droplet digital PCR (ddPCR) is a quantitative assay that requires DNA fragmentation to maximize reaction efficiency. We measured the proportion of mtDNA carrying the “common deletion,” a rare event associated with aging and neurodegeneration. We compared alternative fragmentation methods (sonication and QIAshredder columns) against enzymatic digestion. QIAshredder showed the highest sensitivity when compared to sonication, followed by digestion. QIAshredder and sonication are fragmentation methods that maximize ddPCR quantification for the detection of rare events.