Sequencing strategy for the whole mitochondrial genome resulting in high quality sequences.
Sequencing strategy for the whole mitochondrial genome resulting in high quality sequences.
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DOI：
10.1186/1471-2164-10-139
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发表时间：
2009-03-30
期刊：
影响因子：
4.4
通讯作者：
Parson W
中科院分区：
文献类型：
--
作者：
Fendt L;Zimmermann B;Daniaux M;Parson W
已经证明，可靠且故障安全的测序策略对于线粒体（mt）DNA的高质量分析是强制性的，因为测序和碱基识别过程容易出错。在这里，我们提出了一种高质量，可靠和易于操作的手动程序，用于全mt基因组测序，也适用于没有全自动化过程的实验室。我们扩增了整个线粒体基因组作为两个重叠的PCR片段，每个约8500个碱基的长度。我们开发了一组96个引物，可应用于（手动）96个基于孔的技术，这导致至少双链序列覆盖整个编码区（codR）。这种精心设计的测序策略是直接的，并且允许明确的序列分析和解释，包括有时具有挑战性的现象，例如与法医和临床样品的研究相关的点和长度异质性。
It has been demonstrated that a reliable and fail-safe sequencing strategy is mandatory for high-quality analysis of mitochondrial (mt) DNA, as the sequencing and base-calling process is prone to error. Here, we present a high quality, reliable and easy handling manual procedure for the sequencing of full mt genomes that is also appropriate for laboratories where fully automated processes are not available. We amplified whole mitochondrial genomes as two overlapping PCR-fragments comprising each about 8500 bases in length. We developed a set of 96 primers that can be applied to a (manual) 96 well-based technology, which resulted in at least double strand sequence coverage of the entire coding region (codR). This elaborated sequencing strategy is straightforward and allows for an unambiguous sequence analysis and interpretation including sometimes challenging phenomena such as point and length heteroplasmy that are relevant for the investigation of forensic and clinical samples.
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