Abstract:
:A point mutation of mitochondrial DNA (mtDNA) at nucleotide position 3243 A to G (mt.3243A>G) is involved in many common diseases, including maternally inherited diabetes and deafness (MIDD) and mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes (MELAS). However, the mutant level of mt.3243A>G varies both among individuals and in different organs, tissues, and even cells of single individuals. For detection of this mutation, current methods have limited universality and sensitivity and may be not adequate for a routine clinical test. Here, we develop and evaluate a rapid TaqMan-MGB quantitative real-time PCR (qPCR) method for detecting and quantifying the heteroplasmy level of mt.3243A>G in single-tube analysis. With our method, the sensitivity of detection was as low as 0.1%, but the accuracy of quantification was reliable, down to 4%. All positives could be correctly identified, and the heteroplasmy levels determined by qPCR correlated well with the results from restriction fragment length polymorphism (RFLP) and pyrosequencing assays (r = 0.921~0.973 and 0.972~0.984). In addition, we demonstrated that the urinary sediments, leukocytes, or hair follicles might be ideal templates to detect and quantify the heteroplasmy of mt.3243A>G mutation; however, they should be optimized or retreated for further accurate quantification. Our study should allow rapid and high throughput diagnostic testing and can potentially be used to clarify the association between clinical phenotype and pathogenic mitochondrial mutations derived from various tissues.
journal_name
Biomed Res Intjournal_title
BioMed research internationalauthors
Rong E,Wang H,Hao S,Fu Y,Ma Y,Wang Tdoi
10.1155/2018/1286480subject
Has Abstractpub_date
2018-11-13 00:00:00pages
1286480eissn
2314-6133issn
2314-6141journal_volume
2018pub_type
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