July’s Editor's Choice article is “Design of a digital-PCR assay to quantify fragmented human mitochondrial DNA,” (https://onlinelibrary.wiley.com/doi/10.1002/em.22449) by Alejandro Mosquera, Rebeca Guillén, Fátima Otero, Ignacio Rego-Pérez, Francisco J. Blanco, José Luis Fernández.
Individual mitochondrion contain approximately 2-10 copies of mitochondrial DNA (mtDNA) molecules and the number of mitochondria within a cell can vary greatly based on the energy needs of the cell type and organ. In healthy cells, mtDNA copy number is maintained at a necessary level determined by an as yet unknown mechanism. However, mtDNA copy number and integrity changes have been associated with a multitude of diseases, including Pearson’s syndrome, Kerns-Sayre syndrome, Parkinson’s disease, and Alzheimer’s disease.
Historically, Southern blot hybridization and quantitative PCR (qPCR) are mainstays in the evaluation of the integrity and quantity of mitochondrial DNA (mtDNA) molecules. Aside from being painstakingly laborious, Southern blots required large amounts of DNA and were only semi-quantitative. While much simpler to perform, qPCR reports mtDNA levels in comparison to a selected nuclear gene. More recently, digital PCR (dPCR) allowed for the direct measurement of the absolute mtDNA copy number. Mosquera et al. have developed an assay utilizing dPCR to measure the absolute mtDNA copy number and to quantify the proportion of mtDNA molecules containing DSBs, via a “break-apart” approach.
The assay developed by Mosquera et al. simultaneously amplifies two different small target sequences, ND1 and ND6, located thousands of bases apart in independent microchambers running parallel PCR reactions. Detection of each target is done by hybridization with TaqMan probes labeled with HEX (target 1) and FAM (target 2). Co-localization of HEX and FAM indicate an intact circular or linearized mtDNA. HEX or FAM located alone in a microchamber indicates fragmented mtDNA in both segments connecting the targets. In order to distinguish circular from linear mtDNA, restriction enzyme digests are performed prior to the PCR reaction.
The initial objective of this study was to demonstrate the accuracy of the break apart dPCR assay to detect and quantify fragmented mtDNA. Each sample isolated from healthy human peripheral blood was divided into three reactions: undigested, linearized with a single digest using EagI-HF, and fragmented by a double digest with EagI-HF and BmgBI. Digestion with EagI-HF and BmgBI cleave mtDNA between the two targets in the short and long segments, respectively. By mixing different proportions of linearized and fragmented mtDNA, Mosquera et al. were able to validate the accuracy and sensitively of the assay to identify mtDNA containing 2 or more DSBs, as well as establish the basal level of mtDNA fragmentation of their samples.
By simply altering the third condition of the assay from a double digest to a single digest with BmgBI, Mosquera et al. were able to estimate the proportion of circular, linearized, and fragmented mtDNA in a sample. They determined that the DSBs were indiscriminately dispersed between the long and short segments connecting the targets. The long segment is 1.8 times longer and contained 2.2 times higher fraction of linearized molecules than the short segment.
The next objective of the study by Mosquera et al. was to compare the amount of fragmentation in cellular mtDNA to extracellular mtDNA. Extracellular mtDNA was collected from U2OS cell culture medium. Higher fragmentation was found in the extracellular DNA compared to the cellular DNA at each different confluency. Further digestion of the extracellular mtDNA by EagI-HF and BmgBI did not increase the fragmentation indicating that the majority of extracellular mtDNA was originally fragmented.
The final objective of the study was to observe the effect of ethidium bromide (EtBr) on mtDNA copy number and integrity. EtBr has a high affinity for negatively supercoiled DNA, such as mtDNA. EtBr intercalates into the DNA and inhibits the replication of mtDNA by halting replication by polymerase Gamma. Although the full mechanism is unknown, EtBr is often used to deplete mtDNA from cells. Mosquera et al. discovered that after incubating U2OS cells with EtBr for 24 and 48 hours, the total amount of mtDNA is reduced by nearly 8-fold and 9-fold, respectively. Of the mtDNA remaining after 24 and 48 hours, those subjected to EtBr saw no increase in linear molecules, but a 4-8-fold increase in the proportion of fragmented molecules from 2 or more DSBs. Previous techniques, such as qPCR, would overestimate the number of functional mtDNA molecules remaining.
While this assay design is unable to detect two or more DSBs in the same segment, the probability of two or more breaks located solely in one segment prior to digestion is low if the basal level of breaks is low. Additionally, two or more breaks in the native mtDNA may indicate degradation. The simple assay design by Mosquera et al. can be employed to study conditions and new drugs that can alter mtDNA copy number and fragmentation.