Analysis of mitochondrial DNA point mutation heteroplasmy by ARMS quantitative PCR

Mitochondrial disorders are a group of complex and heterogeneous diseases that may be caused by molecular defects in both the nuclear and mitochondrial genomes. Pathogenic mitochondrial DNA (mtDNA) mutations are usually present in the heteroplasmic form. The degree of mtDNA mutation heteroplasmy (mutation load) varies among different tissues. Manifestation of clinical phenotypes depends on the mutation load in affected tissues. Thus, it is important to quantify the degree of mutation heteroplasmy in various tissues. This unit outlines the design of allele refractory mutation system (ARMS)-based quantitative PCR (qPCR) analysis of three common mtDNA point mutations. This is a cost-effective and sensitive single-step method to simultaneously detect and quantify heteroplasmic mtDNA point mutations. It requires as little as 0.1 ng of total genomic DNA per reaction and can be used to quantify mutation heteroplasmy in various noninvasively obtained tissues such as hair follicles, buccal swab, and urine sediment. Detailed protocols for ARMS primer design and qPCR set up, validation procedures, and cautions in the interpretation of results, as well as advantages and limitations are discussed. ARMS qPCR is an important tool for addressing some of the diagnostic challenges of mitochondrial disease.