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首页> 外文期刊>BMC Biotechnology >Isolation and analysis of high quality nuclear DNA with reduced organellar DNA for plant genome sequencing and resequencing
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Isolation and analysis of high quality nuclear DNA with reduced organellar DNA for plant genome sequencing and resequencing

机译：带有减少的细胞器DNA的高质量核DNA的分离和分析，用于植物基因组测序和重测序

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Background High throughput sequencing (HTS) technologies have revolutionized the field of genomics by drastically reducing the cost of sequencing, making it feasible for individual labs to sequence or resequence plant genomes. Obtaining high quality, high molecular weight DNA from plants poses significant challenges due to the high copy number of chloroplast and mitochondrial DNA, as well as high levels of phenolic compounds and polysaccharides. Multiple methods have been used to isolate DNA from plants; the CTAB method is commonly used to isolate total cellular DNA from plants that contain nuclear DNA, as well as chloroplast and mitochondrial DNA. Alternatively, DNA can be isolated from nuclei to minimize chloroplast and mitochondrial DNA contamination. Results We describe optimized protocols for isolation of nuclear DNA from eight different plant species encompassing both monocot and eudicot species. These protocols use nuclei isolation to minimize chloroplast and mitochondrial DNA contamination. We also developed a protocol to determine the number of chloroplast and mitochondrial DNA copies relative to the nuclear DNA using quantitative real time PCR (qPCR). We compared DNA isolated from nuclei to total cellular DNA isolated with the CTAB method. As expected, DNA isolated from nuclei consistently yielded nuclear DNA with fewer chloroplast and mitochondrial DNA copies, as compared to the total cellular DNA prepared with the CTAB method. This protocol will allow for analysis of the quality and quantity of nuclear DNA before starting a plant whole genome sequencing or resequencing experiment. Conclusions Extracting high quality, high molecular weight nuclear DNA in plants has the potential to be a bottleneck in the era of whole genome sequencing and resequencing. The methods that are described here provide a framework for researchers to extract and quantify nuclear DNA in multiple types of plants.
机译：背景技术高通量测序（HTS）技术通过大大降低测序成本，彻底改变了基因组学领域，使单个实验室对植物基因组进行测序或重新测序变得可行。由于叶绿体和线粒体DNA的拷贝数高，以及酚类化合物和多糖的含量高，因此从植物中获得高质量，高分子量的DNA构成了重大挑战。已经使用多种方法从植物中分离DNA。 CTAB方法通常用于从含有核DNA以及叶绿体和线粒体DNA的植物中分离总细胞DNA。或者，可以从细胞核中分离DNA，以最大程度地减少叶绿体和线粒体DNA污染。结果我们描述了从包括单子叶植物和双子叶植物在内的八种不同植物中分离核DNA的优化方案。这些协议使用核隔离以最小化叶绿体和线粒体DNA污染。我们还开发了一种协议，可以使用定量实时PCR（qPCR）确定相对于核DNA的叶绿体和线粒体DNA拷贝数。我们比较了从核中分离的DNA与用CTAB方法分离的总细胞DNA。正如预期的那样，与用CTAB方法制备的总细胞DNA相比，从细胞核中分离的DNA始终产生具有较少叶绿体和线粒体DNA拷贝的核DNA。该协议将允许在开始植物全基因组测序或重测序实验之前分析核DNA的质量和数量。结论在植物中提取高质量，高分子量的核DNA可能成为全基因组测序和重测序时代的瓶颈。这里介绍的方法为研究人员提供了一个框架，以提取和定量多种植物中的核DNA。

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