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首页> 外文期刊>Electrophoresis: The Official Journal of the International Electrophoresis Society >Human mitochondrial DNA complete amplification and sequencing: a new validated primer set that prevents nuclear DNA sequences of mitochondrial origin co-amplification.
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Human mitochondrial DNA complete amplification and sequencing: a new validated primer set that prevents nuclear DNA sequences of mitochondrial origin co-amplification.

机译：人线粒体DNA完整扩增和测序：一种新的经过验证的引物组，可防止线粒体起源的核DNA序列进行共扩增。

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摘要

To date, there are no published primers to amplify the entire mitochondrial DNA (mtDNA) that completely prevent the amplification of nuclear DNA (nDNA) sequences of mitochondrial origin. The main goal of this work was to design, validate and describe a set of primers, to specifically amplify and sequence the complete human mtDNA, allowing the correct interpretation of mtDNA heteroplasmy in healthy and pathological samples. Validation was performed using two different approaches: (i) Basic Local Alignment Search Tool and (ii) amplification using isolated nDNA obtained from sperm cells by differential lyses. During the validation process, two mtDNA regions, with high similarity with nDNA, represent the major problematic areas for primer design. One of these could represent a non-published nuclear DNA sequence of mitochondrial origin. For two of the initially designed fragments, the amplification results reveal PCR artifacts that can be attributed to the poor quality of the DNA. After the validation, nine overlapping primer pairs to perform mtDNA amplification and 22 additional internal primers for mtDNA sequencing were obtained. These primers could be a useful tool in future projects that deal with mtDNA complete sequencing and heteroplasmy detection, since they represent a set of primers that have been tested for the non-amplification of nDNA.
机译：迄今为止，还没有公开的引物能够扩增整个线粒体DNA（mtDNA），从而完全阻止线粒体来源的核DNA（nDNA）序列的扩增。这项工作的主要目的是设计，验证和描述一组引物，以特异性扩增和测序完整的人类mtDNA，从而可以正确解释健康和病理学样品中的mtDNA异质性。使用两种不同的方法进行验证：（i）基本的局部比对搜索工具，以及（ii）使用通过差异裂解从精细胞中分离得到的nDNA进行扩增。在验证过程中，两个与nDNA高度相似的mtDNA区域代表了引物设计的主要问题区域。其中之一可能代表线粒体来源的未公开的核DNA序列。对于最初设计的两个片段，扩增结果显示了PCR伪影，这可能归因于DNA质量差。验证后，获得了九对重叠的引物对以进行mtDNA扩增，并获得了22种其他内部引物用于mtDNA测序。这些引物可能是将来处理mtDNA完整测序和异质性检测的项目中的有用工具，因为它们代表了一组经过非nDNA扩增测试的引物。

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