Development of a sensitive double TaqMan Probe-based qPCR Angle-Degree method to detect mutation frequencies

• Published in: Mitochondrion 2023-05, Vol.70, p.1-7
• Main Authors: Tomita, Kazuo, Indo, Hiroko P., Sato, Tomoaki, Tangpong, Jitbanjong, Majima, Hideyuki J.
• Format: Article
• Language: English
• Subjects: DNA, Mitochondrial - genetics; Double TaqMan probes; Humans; MELAS; MELAS Syndrome - genetics; Mitochondria; mitochondrial DNA; Mutation; Mutation Rate; Stroke
• ISSN: 1567-7249; 1872-8278
• DOI: 10.1016/j.mito.2023.02.010

•• The mtDNA mutation frequency could be calculated by qPCR using the FAM and VIC probes.•• This method was used to draw a standard curve by applying the angle-degree method.•• The accuracy of the mutation frequency obtained by this method (2.4 ± 1. %) was higher vs. the existing methods.•• This method can also be applied to obtain other mutation frequencies in DNA other than mtDNA, e.g., in the context of diabetes, aging, cancer, and neurodegenerative diseases. We designed a method to examine the mutation frequencies of the A3243G mutation of mitochondrial DNA (mtDNA) in patients with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome. We performed a qPCR assay using the FAM and VIC TaqMan probes, which detect the 3243G (mutated) and 3243A (wild-type) sequences of mtDNA, respectively. The results obtained by “degree” in a series of differential mutation frequencies were used to plot a standard curve of the mutation frequency. The standard curve was then applied for qPCR assays of the desired samples. The standard deviation (%) of the samples calculated using the standard curve for the TaqMan probe was 2.4 ± 1.5%. This method could be used to examine mutation frequencies in the context of diabetes, aging, cancer, and neurodegenerative diseases.