Extremely rare mitochondrial DNA deletions associated with aging can be accurately detected with Droplet Digital PCR

September 4, 2013

A study published today in Aging Cell identifies a new tool to accurately analyze extremely rare mitochondrial DNA (mtDNA) deletions associated with a range of diseases and disorders as well as aging. This approach, which relies on Droplet Digital PCR (ddPCR) technology, will help researchers explore mtDNA deletions as potential disease biomarkers.

The accumulation of mtDNA mutations is associated with aging, , and cancer. However, methods to probe the underlying mechanisms behind this mutagenesis have been limited by their inability to accurately quantify and characterize new deletion events, which may occur at a frequency as low as one deletion event per 100 million mitochondrial genomes in normal tissue. To address these limitations, researchers at the Seattle, Washington-based Fred Hutchinson Cancer Research Center developed a ddPCR-based assay known as "Digital Deletion Detection" (3D) that allows for the high-resolution analysis of these rare deletions.

"It is incredibly difficult to study mtDNA mutations, let alone deletions, within the ," said Dr. Jason Bielas, Assistant Member of the Public Health Sciences Division at the Fred Hutchinson Cancer Research Center and lead author of the study. "Our 3D assay shows significant improvement in specificity, sensitivity, and accuracy over conventional methods such as those that rely on real-time PCR."

Bielas added, "The increase in throughput afforded by droplet digital PCR shortened the analysis of deletion events to days compared to months using previous digital PCR methods. Without the technology, we could not have made this discovery."

At the center of the study was 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 formation and expansion, and their role in aging. Droplet digital PCR's high throughput and increased sensitivity will also allow Bielas' lab to target other low-level disease-causing mtDNA deletions in skeletal muscle, brain tissue, and blood.

Explore further: New biomarker could reveal Alzheimer's disease years before onset

Related Stories

New biomarker could reveal Alzheimer's disease years before onset

August 14, 2013
A study published today reported the identification of what may be the earliest known biomarker associated with the risk of developing Alzheimer's disease (AD). The results suggest that this novel potential biomarker is present ...

Rare mitochondrial mutations—maybe not so rare?

June 8, 2013
French scientists have discovered that supposedly rare mutations in the mitochondria, the 'power plants' of human cells responsible for creating energy, account for more than 7% of patients with a mitochondrial disease manifesting ...

Mechanism that allows bacteria to infect plants may inspire cure for eye disease

August 4, 2013
By borrowing a tool from bacteria that infect plants, scientists have developed a new approach to eliminate mutated DNA inside mitochondria—the energy factories within cells. Doctors might someday use the approach to treat ...

Study sheds new light on role of genetic mutations in colon cancer development

June 7, 2012
In exploring the genetics of mitochondria – the powerhouse of the cell – researchers at Fred Hutchinson Cancer Research Center have stumbled upon a finding that challenges previously held beliefs about the role ...

Is short stature associated with a 'shortage' of genes?

November 23, 2011
New research sifts through the entire genome of thousands of human subjects to look for genetic variation associated with height. The results of the study, published by Cell Press in the December issue of the American Journal ...

Recommended for you

Scientists provide insight into genetic basis of neuropsychiatric disorders

July 21, 2017
A study by scientists at the Children's Medical Center Research Institute at UT Southwestern (CRI) is providing insight into the genetic basis of neuropsychiatric disorders. In this research, the first mouse model of a mutation ...

South Asian genomes could be boon for disease research, scientists say

July 18, 2017
The Indian subcontinent's massive population is nearing 1.5 billion according to recent accounts. But that population is far from monolithic; it's made up of nearly 5,000 well-defined sub-groups, making the region one of ...

Mutant yeast reveals details of the aberrant genomic machinery of children's high-grade gliomas

July 18, 2017
St. Jude Children's Research Hospital biologists have used engineered yeast cells to discover how a mutation that is frequently found in pediatric brain tumor high-grade glioma triggers a cascade of genomic malfunctions.

Late-breaking mutations may play an important role in autism

July 17, 2017
A study of nearly 6,000 families, combining three genetic sequencing technologies, finds that mutations that occur after conception play an important role in autism. A team led by investigators at Boston Children's Hospital ...

Newly identified genetic marker may help detect high-risk flu patients

July 17, 2017
Researchers have discovered an inherited genetic variation that may help identify patients at elevated risk for severe, potentially fatal influenza infections. The scientists have also linked the gene variant to a mechanism ...

Newly discovered gene variants link innate immunity and Alzheimer's disease

July 17, 2017
Three new gene variants, found in a genome wide association study of Alzheimer's disease (AD), point to the brain's immune cells in the onset of the disorder. These genes encode three proteins that are found in microglia, ...

0 comments

Please sign in to add a comment. Registration is free, and takes less than a minute. Read more

Click here to reset your password.
Sign in to get notified via email when new comments are made.