Li Zhigang, Parris Stephen, Saski Christopher A
Department of Plant and Environmental Sciences, College of Agriculture, Forestry and Life Sciences, Clemson University, Clemson, SC 29634 USA.
Plant Methods. 2020 Mar 14;16:38. doi: 10.1186/s13007-020-00579-4. eCollection 2020.
High-molecular-weight and pure DNA is crucial for high-quality results from 3rd generation DNA Analyzers and optical mapping technologies. Conventional nuclei isolation methods for preparing high-molecular-weight genomic DNA from plant tissues include the preparation of protoplasts or embedding nuclei in an agarose matrix with subsequent manipulations via electro-elution or pulsed-field gel electrophoresis.
In this method, plant nuclei are isolated by physically grinding tissues and reconstituting the intact nuclei in a unique Nuclear Isolation Buffer (NIB). The plastid DNAs are released from organelles and eliminated with an osmotic buffer by washing and centrifugation. The purified nuclei are then lysed and further cleaned by organic extraction, and the genomic DNA is precipitated with a high concentration of CTAB. The highly pure, high molecular weight gDNA is extracted from the nuclei, dissolved in a high pH buffer, allowing for stable long-term storage.
This method is unique and avoids the use of embedding in agarose, which dramatically reduces time (4-8 h versus days), complexity, and materials cost. This procedure can be used on essentially any plant species and tissue stage. Here we describe a case study and a simple method to rapidly prepare high molecular weight gDNA from Upland cotton, blackgrass, and strawberry suitable for single-molecule sequencing.
高分子量且纯净的DNA对于第三代DNA分析仪和光学图谱技术获得高质量结果至关重要。从植物组织中制备高分子量基因组DNA的传统细胞核分离方法包括制备原生质体或将细胞核包埋在琼脂糖基质中，随后通过电洗脱或脉冲场凝胶电泳进行操作。
在该方法中，通过物理研磨组织分离植物细胞核，并在一种独特的核分离缓冲液（NIB）中重构完整的细胞核。质体DNA从细胞器中释放出来，并通过洗涤和离心用渗透缓冲液去除。然后裂解纯化的细胞核，通过有机萃取进一步纯化，并用高浓度的十六烷基三甲基溴化铵沉淀基因组DNA。从细胞核中提取出高纯度、高分子量的基因组DNA，溶解在高pH缓冲液中，可实现长期稳定保存。
该方法独具特色，避免了琼脂糖包埋的使用，显著减少了时间（从数天缩短至4 - 8小时）、操作复杂性和材料成本。该程序基本上可用于任何植物物种和组织阶段。在此，我们描述了一个案例研究以及一种从陆地棉、黑麦草和草莓中快速制备适用于单分子测序的高分子量基因组DNA的简单方法。
