Taylor Sean D, Ericson Nolan G, Burton Joshua N, Prolla Tomas A, Silber John R, Shendure Jay, Bielas Jason H
Translational Research Program, Public Health Sciences Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave, Seattle, WA, 98109, USA.
Aging Cell. 2014 Feb;13(1):29-38. doi: 10.1111/acel.12146. Epub 2013 Sep 11.
Due largely to the inability to accurately quantify and characterize de novo deletion events, the mechanisms underpinning the pathogenic expansion of mtDNA deletions in aging and neuromuscular disorders remain poorly understood. Here, we outline and validate a new tool termed 'Digital Deletion Detection' (3D) that allows for high-resolution analysis of rare deletions occurring at frequencies as low as 1 × 10(-8) . 3D is a three-step process that includes targeted enrichment for deletion-bearing molecules, single-molecule partitioning of genomes into thousands of droplets for direct quantification via droplet digital PCR, and breakpoint characterization using massively parallel sequencing. Using 3D, we interrogated over 8 billion mitochondrial genomes to analyze the age-related dynamics of mtDNA deletions in human brain tissue. We demonstrate that the total deletion load increases with age, while the total number and diversity of unique deletions remain constant. Our data provide support for the hypothesis that expansion of pre-existing mutations is the primary factor contributing to age-related accumulation of mtDNA deletions.
很大程度上由于无法准确量化和表征新生缺失事件，衰老和神经肌肉疾病中线粒体DNA（mtDNA）缺失致病性扩增的潜在机制仍知之甚少。在此，我们概述并验证了一种名为“数字缺失检测”（3D）的新工具，它能够对频率低至1×10⁻⁸的罕见缺失进行高分辨率分析。3D是一个三步过程，包括对携带缺失的分子进行靶向富集、将基因组单分子分配到数千个液滴中以通过液滴数字PCR进行直接定量，以及使用大规模平行测序对断点进行表征。利用3D，我们检测了超过80亿个线粒体基因组，以分析人类脑组织中mtDNA缺失与年龄相关的动态变化。我们证明，总缺失负荷随年龄增长而增加，而独特缺失的总数和多样性保持不变。我们的数据支持以下假说：预先存在的突变的扩增是导致与年龄相关的mtDNA缺失积累的主要因素。
