New technology accurately quantifies rare mtDNA deletions

The accumulation of mtDNA mutations is closely related to aging, neuromuscular diseases and cancer. However, current methods cannot accurately quantify and identify new deletion events because of their very low frequency, with about one deletion event per 100 million mitochondrial genomes in normal tissues. To address these limitations, the researchers developed an analysis method based on ddPCR to analyze these rare deletions with high resolution.

Researchers at the Fred Hutchinson Cancer Research Center recently discovered a new tool that can accurately analyze extremely rare mitochondrial DNA deletions associated with a variety of diseases and aging. This result was published online in "Aging Cell" magazine.

The accumulation of mtDNA mutations is closely related to aging, neuromuscular diseases and cancer. However, current methods cannot accurately quantify and identify new deletion events because of their very low frequency, with about one deletion event per 100 million mitochondrial genomes in normal tissues. To address these limitations, the researchers developed an analysis method based on ddPCR to analyze these rare deletions with high resolution. They call this method "Digital Deletion Detection (Digital Deletion Detection)", referred to as 3D.

The corresponding author of the article, Dr. Jason Bielas, said: "It is extremely difficult to study mtDNA mutations, not to mention deletions. Our 3D detection has significantly improved specificity, sensitivity and accuracy compared to traditional methods. The increased throughput brought by digital digital PCR has shortened the analysis time from months to days. Without this technology, we could not make this discovery. "

Dr. Bielas refers to Bio-Rad's QX100? DdPCR system. Using this system, the research team analyzed 8 billion human brain mtDNA genomes and identified more than 100,000 genomes with deletions. They found that, contrary to popular belief, most increases in mtDNA deletions are not caused by new deletions, but by amplification of previous deletions. They speculate that the amplification of existing mutations should be the main factor that causes the accumulation of mtDNA deletions.

3D is a novel three-step process that includes enrichment of molecules with deletions; the genome is divided into single-molecule droplets and quantified by ddPCR; the breakpoint is determined using next-generation sequencing.

After the enrichment is completed, the molecular concentration in the droplets is adjusted by the QX100 system so that most droplets do not contain mutant genomes, and a few have only one. This process allows each deletion to be amplified without bias, without introducing errors like qPCR.

After amplification, Bielas and his team used ddPCR to determine the absolute concentration of the mutant molecule. Based on the relationship between droplet fluorescence and amplicon size, they were able to identify the size and complexity of rare deletions in brain samples (whether it was the amplification of a few clones or a large number of random deletions).

The researchers believe that 3D detection, as an important new tool, will help to better study the mechanism of deletion formation and amplification, as well as their role in aging. The high throughput and high sensitivity of droplet digital PCR also allowed Dr. Bielas' laboratory to target other low-level pathogenic mtDNA deletions in skeletal muscle, brain tissue, and blood.

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