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

Kazuo Tomita; Hiroko P Indo; Tomoaki Sato; Jitbanjong Tangpong; Hideyuki J Majima

doi:10.1016/j.mito.2023.02.010

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

Mitochondrion. 2023 May:70:1-7. doi: 10.1016/j.mito.2023.02.010. Epub 2023 Feb 24.

Authors

Kazuo Tomita¹, Hiroko P Indo², Tomoaki Sato³, Jitbanjong Tangpong⁴, Hideyuki J Majima⁵

Affiliations

¹ Department of Maxillofacial Radiology, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan; Department of Applied Pharmacology, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan.
² Department of Maxillofacial Radiology, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan; Amanogawa Galactic Astronomy Research Center (AGARC), Kagoshima University Graduate School of Sciences and Engineering, 1-21-35 Korimoto, Kagoshima 890-0065, Japan. Electronic address: hindoh@dent.kagoshima-u.ac.jp.
³ Department of Applied Pharmacology, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan.
⁴ School of Allied Health Sciences, Walailak University, Nakhon Si Thammarat 80160, Thailand; Research Excellence Center for Innovation and Health Products (RECIHP), School of Allied Health Sciences, Walailak University, Nakhon Si Thammarat 80160, Thailand.
⁵ Department of Maxillofacial Radiology, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan; Amanogawa Galactic Astronomy Research Center (AGARC), Kagoshima University Graduate School of Sciences and Engineering, 1-21-35 Korimoto, Kagoshima 890-0065, Japan; School of Allied Health Sciences, Walailak University, Nakhon Si Thammarat 80160, Thailand; Research Excellence Center for Innovation and Health Products (RECIHP), School of Allied Health Sciences, Walailak University, Nakhon Si Thammarat 80160, Thailand; Center of Excellence Research for Melioidosis and Microorganisms, Walailak University, Thasala, Nakhon Si Thammarat 80161, Thailand. Electronic address: k0941761@kadai.jp.

PMID: 36841519
DOI: 10.1016/j.mito.2023.02.010

Abstract

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.

Keywords: Double TaqMan probes; MELAS; Mitochondria; Mutation; Mutation rate; mitochondrial DNA.

MeSH terms

DNA, Mitochondrial / genetics
Humans
MELAS Syndrome* / genetics
Mutation
Mutation Rate
Stroke*

Substances

DNA, Mitochondrial