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In addition to the methylation of genomic DNA that occurs in the nucleus, which can be used as an epigenetic marker, the methylation of mitochondrial DNA (mtDNA) has gradually been paid more attention. In some studies on mitochondria, varying degrees of methylation modification (less than a quarter of the degree of methylation in the nucleus) have been detected. Mitochondrial function is related to the pathophysiological processes of neurological diseases and some metabolic diseases. For example, mitochondrial dysfunction plays a key role in cancer development and related treatment responses. Mitochondrial dysfunction in a wide range of human malignancies indicates that targeting mitochondria is still a promising way to develop new cancer treatment strategies. The data shows that the mitochondrial genome is largely methylated at non-CpG sites.
The circular double-stranded structure of the mitochondrial genome is similar to that of the bacterial genome. Different from the nuclear genome is its complex secondary and tertiary structure, which will make the detection of mitochondrial DNA methylation more difficult. The secondary structure of mitochondrial DNA may prevent the bisulfite conversion step, leading to false-positive methylation values.
Fig 1. Map of the human mitochondrial genome. (Mechta M, et al. 2017)
The current mtDNA detection strategy is mainly the bisulfite method. However, unlike nuclear genomes, other methods can be used to unravel the secondary and tertiary structure of mtDNA. Accurate fragmentation is the key to the detection of mitochondrial DNA methylation, and the optimization of this step can ensure high quality and reliable methylation value. Avoid the low bisulfite conversion efficiency due to the circular ring structure of mtDNA, which will cause the increase of methylation measurements.
|Mitochondrial DNA Extraction||Use the classic DNA extraction method of organelles. Collect the minimum amount of nucleic acid required for bisulfite modification (2μg concentration).|
|Linearization||Enzyme digestion and ultrasonic treatment were used to achieve the best linearization. The application of enzyme digestion (BamHI restriction endonuclease) can break the loop of mitochondrial circular DNA, further ultrasonic treatment can make it fragment, unravel the secondary and tertiary structure, and facilitate the subsequent conversion of bisulfite.|
|Bisulfite Conversion||Use the sulfite conversion kit.|
|MeIP||Purification and enrichment using immunological methods.|
|Detection||Microarray hybridization detection.|
Fig 2. Mitochondrial DNA Methylation Detection Service.
CD Genomics provides high-quality mitochondrial DNA methylation microarray screening services. We have a well-equipped experimental platform, a large and professional team of scientists, and we are committed to cooperating with researchers from all over the world to meet the needs of our customers. Customers can contact our employees directly and provide timely feedback on their queries. If you are interested in our one-stop solution service, please contact us for more detailed information.