Yen Hsiu-Chuan, Li Shiue-Li, Hsu Wei-Chien, Tang Petrus
Department and Graduate Institute of Medical Biotechnology and Laboratory Sciences, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan.
Department of Public Health and Parasitology, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan.
PLoS One. 2014 Mar 24;9(3):e92817. doi: 10.1371/journal.pone.0092817. eCollection 2014.
High-sensitivity and high-throughput mutation detection techniques are useful for screening the homoplasmy or heteroplasmy status of mitochondrial DNA (mtDNA), but might be susceptible to interference from nuclear mitochondrial DNA sequences (NUMTs) co-amplified during polymerase chain reaction (PCR). In this study, we first evaluated the platform of SURVEYOR Nuclease digestion of heteroduplexed DNA followed by the detection of cleaved DNA by using the WAVE HS System (SN/WAVE-HS) for detecting human mtDNA variants and found that its performance was slightly better than that of denaturing high-performance liquid chromatography (DHPLC). The potential interference from co-amplified NUMTs on screening mtDNA heteroplasmy when using these 2 highly sensitive techniques was further examined by using 2 published primer sets containing a total of 65 primer pairs, which were originally designed to be used with one of the 2 techniques. We confirmed that 24 primer pairs could amplify NUMTs by conducting bioinformatic analysis and PCR with the DNA from 143B-ρ0 cells. Using mtDNA extracted from the mitochondria of human 143B cells and a cybrid line with the nuclear background of 143B-ρ0 cells, we demonstrated that NUMTs could affect the patterns of chromatograms for cell DNA during SN-WAVE/HS analysis of mtDNA, leading to incorrect judgment of mtDNA homoplasmy or heteroplasmy status. However, we observed such interference only in 2 of 24 primer pairs selected, and did not observe such effects during DHPLC analysis. These results indicate that NUMTs can affect the screening of low-level mtDNA variants, but it might not be predicted by bioinformatic analysis or the amplification of DNA from 143B-ρ0 cells. Therefore, using purified mtDNA from cultured cells with proven purity to evaluate the effects of NUMTs from a primer pair on mtDNA detection by using PCR-based high-sensitivity methods prior to the use of a primer pair in real studies would be a more practical strategy.
高灵敏度和高通量突变检测技术有助于筛查线粒体DNA（mtDNA）的同质性或异质性状态，但在聚合酶链反应（PCR）过程中可能容易受到共扩增的核线粒体DNA序列（NUMTs）的干扰。在本研究中，我们首先评估了用于检测人类mtDNA变异的SURVEYOR核酸酶消化异源双链DNA并随后使用WAVE HS系统（SN/WAVE-HS）检测切割DNA的平台，发现其性能略优于变性高效液相色谱法（DHPLC）。我们使用2组已发表的引物对（共65对引物）进一步研究了在使用这两种高灵敏度技术筛查mtDNA异质性时共扩增NUMTs的潜在干扰，这2组引物对最初设计用于这两种技术之一。通过生物信息学分析以及使用来自143B-ρ0细胞的DNA进行PCR，我们确认有24对引物可扩增NUMTs。使用从人类143B细胞线粒体中提取的mtDNA以及具有143B-ρ0细胞核背景的细胞杂交系，我们证明在mtDNA的SN-WAVE/HS分析过程中，NUMTs会影响细胞DNA的色谱图模式，从而导致对mtDNA同质性或异质性状态的错误判断。然而，我们仅在所选的24对引物对中的2对中观察到这种干扰，而在DHPLC分析过程中未观察到这种影响。这些结果表明，NUMTs会影响低水平mtDNA变异的筛查，但可能无法通过生物信息学分析或从143B-ρ0细胞中扩增DNA来预测。因此，在实际研究中使用引物对之前，使用来自已证实纯度的培养细胞的纯化mtDNA来评估引物对中的NUMTs对基于PCR的高灵敏度方法检测mtDNA的影响将是一种更实用的策略。
