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Extremely rare mitochondrial DNA deletions associated with aging can be accurately detected with Droplet Digital PCR
Date:9/4/2013

ad.com/en-us/product/digital-pcr-technology/qx100-droplet-digital-pcr-system?tab=Description&WT.mc_id=pr-GXD-ww-qx100ddpcr_BielasAgingCellQX100_20130904_Zh8eE" target="_blank">Bio-Rad Laboratories' QX100 ddPCR system. Using the QX100 system, Bielas and his team analyzed eight billion human brain mtDNA genomes and identified more than 100,000 genomes with a deletion. They discovered that, contrary to popular belief, the majority of the increase in mtDNA deletions was not caused by new deletions but rather by the expansion of previous deletions. They hypothesized that the expansion of pre-existing mutations should be considered as the primary factor contributing to age-related accumulation of mtDNA deletions.

How the 3D Assay Works

3D is a novel three-step process that includes enrichment for deletion-bearing molecules, single-molecule partitioning of genomes into droplets for direct quantification via ddPCR, and breakpoint characterization using next-generation sequencing.

Once the enrichment process is completed using methods previously developed by Bielas and colleagues, the concentration of molecules within the droplets is adjusted by using the QX100 system so that the majority of droplets contain no mutant genomes while a small fraction contain only one. This process allows each deletion to be amplified without bias and without introducing the artifacts that are common in qPCR.

Following amplification, deletions can be analyzed using ddPCR to determine the absolute concentration of mutated molecules. Using the relationship between droplet fluorescence and amplicon size, Bielas and his team were able to characterize the size and complexity (whether they were a result of a few clonal expansions or a large collection of random deletions) of rare mitochondrial deletions in human brain samples.

The 3D assay provides an important new tool that will allow researchers to better study the mechanisms of deletion forma
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Contact: Ken Li
kli@chempetitive.com
312-532-4675
Chempetitive Group
Source:Eurekalert

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