Heteroplasmy Detection of Mitochondrial DNA A3243G Mutation Using Quantitative Real-Time PCR Assay Based on TaqMan-MGB Probes.

Heteroplasmy Detection of Mitochondrial DNA A3243G Mutation Using Quantitative Real-Time PCR Assay Based on TaqMan-MGB Probes. Biomed Res Int. 2018;2018:1286480 Authors: Rong E, Wang H, Hao S, Fu Y, Ma Y, Wang T 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 ...
Source: Biomed Res - Category: Research Authors: Tags: Biomed Res Int Source Type: research