Clinical Pathology: General Principles, Lab Management, Clinical Chemistry, Immunology & Histocompatibility, Genetic Testing

• Traditionally, estimating mitochondrial DNA (mtDNA) copy number relied on Southern blotting approaches. This involves using two labeled probes, one specific for mtDNA and one specific for a nuclear gene, usually a housekeeping gene, such as gluceraldehyde diphosphate dehydrogenase (GAPDH) or β-globulin. In this method, mtDNA is linearized after digestion with an endonuclease enzyme, such as PvuII, and then transferred to a nylon membrane. After autoradiography, the intensity of bands corresponding to mtDNA and the nuclear gene are measured by densitometry. This method is very labor intensive, time consuming, and expensive. Furthermore, it is semi-quantitative and only allows an estimate of mtDNA content.

• mtDNA can also be quantified by polymerase chain reaction (PCR). Competitive PCR is one approach, which involves introduction of a serially diluted competitive template into the tissue extract. mtDNA quantitation is achieved by radiometric comparison of the relative amounts of the two products.

• In recent years, the development of quantitative real-time PCR (qPCR) technology has significantly improved the estimation of mtDNA copy number in clinical specimens, such as blood and tissue. qPCR assays are extremely sensitive and can detect only a few copies, or even a single copy, of target DNA in a clinical specimen.

• Two chemistries, TaqMan and SYBR green, are most commonly used. Assays based on TaqMan chemistry have the advantage of being capable of simultaneously quantifying more than one target in a sample. SYBR-based qPCR assays, although more convenient than TaqMan-based assays, are less accurate because SYBR green can bind to DNA molecules other than the target. Therefore, highly optimized PCR conditions are a prerequisite for successful application of this approach.

• Two approaches are used to calculate mtDNA in qPCR assays. (1) The first calculation uses a standard curve, in which known amounts of DNA are used to construct the standard curve from which the copy number of the unknown sample is delineated. (2) Another approach uses relative quantification, in which the relative copy number of target DNA to a reference DNA, amplified simultaneously, is calculated. The advantage of the latter method is that it obviates the need for a standard curve. However, when the latter method is used, it is imperative to ensure that the efficiencies of target and reference amplification are very similar.



 
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