Quantitative Analysis of Somatic Mitochondrial DNA Mutations by Single-Cell Single-Molecule PCR
Quantitative Analysis of Somatic Mitochondrial DNA Mutations by Single-Cell Single-Molecule PCR
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DOI：
10.1007/978-1-59745-521-3_21
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发表时间：
2009-01-01
期刊：
影响因子：
--
通讯作者：
Khrapko, Konstantin
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文献类型：
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作者：
Kraytsberg, Yevgenya;Bodyak, Natalya;Khrapko, Konstantin
线粒体基因组完整性是体细胞线粒体遗传学中的一个重要问题。定量方法的发展对体细胞线粒体遗传学是必不可少的，因为定量研究需要表征异质性和突变过程，以及它们对表型发展的影响。定量研究包括鉴定和测量致病性和非致病性克隆突变的负荷，筛选线粒体基因组中的突变，以确定突变谱并表征正在进行的突变过程。单分子PCR （smPCR）已被证明是一种有效的方法，可应用于所有领域的定量研究。与传统的基于载体的克隆技术相比，它具有明显的优势，避免了众所周知的PCR相关的人工产物，如引入人工突变、优先等位基因扩增和“跳跃”PCR。smPCR是一种简单而可靠的方法，即使涉及线粒体全基因组（mtWG）分析，也可以有效地用于逐分子突变分析。本章描述了smPCR方法的主要特点，并提供了其在单细胞分析中的三个应用实例：用于缺失定量的双工smPCR，用于克隆点突变定量的smPCR克隆，以及用于全基因组测序（mt:WGS）的smPCR克隆。
Mitochondrial genome integrity is ail important issue in somatic mitochondrial genetics. Development of quantitative methods is indispensable to somatic mitochondrial genetics as quantitative Studies arc required to characterize heteroplasmy and mutation processes, as well as their effects oil phenotypic developments. Quantitative studies include the identification and measurement of the load of pathogenic and non-pathogenic clonal Mutations, screening mitochondrial genomes for mutations in order to determine the mutation spectra and characterize an Ongoing Mutation process. Single-molecule PCR (smPCR) has been shown to be an effective method that can be applied to all areas of quantitative studies. It has distinct advantages over conventional vector-based cloning techniques avoiding the well-known PCR-related artifacts Such as the introduction of artificial mutations, preferential allelic amplifications, and "jumping" PCR. smPCR is a straightforward and robust method, which can be effectively used for molecule-by-molecule mutational analysis, even when mitochondrial whole genome (mtWG) analysis is involved. This chapter describes the key features of the smPCR method and provides three examples of its applications in single-cell analysis: di-plex smPCR for deletion quantification, smPCR cloning for clonal point mutation quantification, and smPCR cloning for whole genome sequencing (mt:WGS).