QUANTITATIVE-DETERMINATION OF DELETED MITOCHONDRIAL-DNA RELATIVE TO NORMAL DNA IN PARKINSONIAN STRIATUM BY A KINETIC PCR ANALYSIS
QUANTITATIVE-DETERMINATION OF DELETED MITOCHONDRIAL-DNA RELATIVE TO NORMAL DNA IN PARKINSONIAN STRIATUM BY A KINETIC PCR ANALYSIS
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DOI：
10.1016/0006-291x(90)90698-m
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发表时间：
1990-10-30
影响因子：
3.1
通讯作者：
MIZUNO, Y
中科院分区：
文献类型：
--
作者：
OZAWA, T;TANAKA, M;MIZUNO, Y
在帕金森病患者的纹状体中积累了大量的线粒体DNA，但当聚合酶链反应的循环次数增加时，在对照纹状体中也可检测到相同的缺失的线粒体DNA。为了区分这些病理和生理状况，我们通过测量α-mtDNA的掺入来定量分析缺失的mtDNA与正常mtDNA的比例。[32 P]脱氧胞嘧啶三磷酸掺入mtDNA片段。为了估计缺失的mtDNA与正常mtDNA的摩尔比，将放射性通过每个片段大小标准化。通过绘制归一化放射性对PCR扩增循环的曲线图，获得具有不同斜率的直线。通过外推到零扩增的直线，估计帕金森病患者纹状体原始样本中突变mtDNA与正常mtDNA的比例约为1.5%。5%，比ca.对照纹状体中为0.3%。这些结果表明，当缺失的mtDNA与正常mtDNA的比例超过正常受试者的10倍阈值时，突变mtDNA表现为帕金森病。
Deleted mitochondrial DNA (mtDNA) was accumulated in the parkinsonian striatum, but the same deleted mtDNA was also detectable in the control striatum when cycles of polymeras chain reaction were increased. To discriminate between these pathological and physiological conditions, we quantitatively analyzed the proportion of deleted mtDNA to normal mtDNA by measuring the incorporation of .alpha.-[32P]deoxycytosine triphosphate into mtDNA fragments by using a laser image analyzer. To estimate the molar ratio of the deleted mtDNA to normal mtDNA, the radioactivity was normalized by each fragment size. By plotting logarithms of normalized radioactivities against PCR amplification cycles, straight lines were obtained with different slopes. By extrapolation of the line to the zero amplification, the proportion of mutant mtDNA to normal mtDNA in the original sample from the parkinsonian striatum was estimated to be ca. 5%, which was at least ten times higher than the proportion of ca. 0.3% in the control striatum. These results indicate that phenotype of the mutant mtDNA as Parkinson''s disease is expressed when the proportion of deleted mtDNA to normal mtDNA exceeds a threshold of ten times higher value than in the normal subjects.