Biffi Stefania, Bortot Barbara, Carrozzi Marco, Severini Giovanni Maria
Department of Molecular Medicine and Laboratory, Optical Imaging Laboratory, CBM Scrl, Area Science Park, Trieste, Italy.
Diagn Mol Pathol. 2011 Jun;20(2):117-22. doi: 10.1097/PDM.0b013e3181efe2c6.
In many mitochondrial diseases, different clinical manifestations are related to tissue-specific distribution of mutated mitochondrial DNA (mtDNA). In this study, we describe an assay for the determination of mutated mtDNA copy number in small clinical samples, using standard polymerase chain reaction (PCR) followed by SYBR Green real-time allelic-specific PCR [amplification refractory mutation system-quantitative PCR (ARMS-qPCR)]. To assess the degree of heteroplasmy in a patient harboring 2 cosegregating mtDNA mutations (4415A>G and 9922A>C) starting from picogram amounts of DNA, we first amplified the mutated target sequence by standard PCR, and then analyzed it by real-time ARMS-qPCR. To validate this method, we analyzed by real-time ARMS-qPCR the PCR amplification products derived from different mixtures containing known proportions of mutant and wild-type cloned mtDNA fragments. The correlation coefficient of 0.994 between expected and observed values for the percentage of mutant A4415G confirms that the relative proportion of mutated and wild-type mtDNA was maintained after the first PCR amplification. This method allows the precise quantification of heteroplasmic mutations in DNA samples extracted from hairs, urine, small stomach biopsies, and, more importantly, single-muscle fiber, with a limit of detection close to 0.5%. This nested real-time ARMS-PCR represents a rapid, efficient, and less expensive method for the detection and quantification of heteroplasmic mutant mtDNA, even in very small clinical samples.
在许多线粒体疾病中，不同的临床表现与突变的线粒体DNA（mtDNA）的组织特异性分布有关。在本研究中，我们描述了一种用于测定小临床样本中突变mtDNA拷贝数的检测方法，该方法采用标准聚合酶链反应（PCR），随后进行SYBR Green实时等位基因特异性PCR[扩增阻滞突变系统定量PCR（ARMS-qPCR）]。为了从皮克量的DNA开始评估携带2个共分离的mtDNA突变（4415A>G和9922A>C）的患者的异质性程度，我们首先通过标准PCR扩增突变的靶序列，然后通过实时ARMS-qPCR进行分析。为了验证该方法，我们通过实时ARMS-qPCR分析了来自含有已知比例的突变型和野生型克隆mtDNA片段的不同混合物的PCR扩增产物。突变型A4415G百分比的预期值与观察值之间的相关系数为0.994，这证实了在第一次PCR扩增后，突变型和野生型mtDNA的相对比例得以维持。该方法能够对从毛发、尿液、小胃活检组织，更重要的是单根肌纤维中提取的DNA样本中的异质性突变进行精确定量，检测限接近0.5%。这种巢式实时ARMS-PCR代表了一种快速、高效且成本较低的方法，用于检测和定量异质性突变mtDNA，即使是在非常小的临床样本中。
