State Key Laboratory of Agricultural Microbiology, Hubei Hongshan Laboratory, Hubei Key Lab of Plant Pathology, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, China.
Shenzhen Institute of Nutrition and Health, Huazhong Agricultural University, Wuhan, China.
Curr Protoc. 2021 Nov;1(11):e289. doi: 10.1002/cpz1.289.
Amplification of genomic DNA fragments by PCR is necessary for plant molecular biology approaches such as genotyping. While this is a routine molecular technique in a modern laboratory, there are still significant hurdles when analyzing a large number of samples or collecting and storing samples while in the field. Because PCR amplification directly from plant tissue is often unsuccessful due to various inhibitors, genomic DNA purification is usually required, which involves laborious and time-consuming procedures or costly materials, particularly when using commercial kits. These undermine scalability and use in less-equipped settings. In addition, plant tissues and purified DNA need to be stored under proper conditions to avoid degradation. Here, we describe a low-cost, high-throughput PCR method to amplify genomic DNA fragments from plant tissue pounded to cellulose-based filter paper without the need for DNA purification or special equipment for sample storage. In this protocol, a small punch of plant tissue is pounded to a commercially available or homemade DNA storage card and directly placed into a PCR mixture containing Tween-20, a non-ionic detergent, directly followed by PCR. We also describe the steps to prepare a homemade DNA storage card, which is easy to make and can be stored with plant tissue at room temperature for a long time without any special equipment, allowing us to test the same sample multiple times. We have used this method in at least eleven plant species, including arabidopsis, tomato, soybean, potato, cotton, and rice. Altogether, our method decreases labor and cost, thereby increasing throughput and making plant DNA-based molecular diagnostic assays accessible to resource-limited settings, including classrooms, and facilitating sample collection in the field. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Making a homemade cellulose-based DNA storage card Basic Protocol 2: Pounding plant tissue on a DNA storage card Basic Protocol 3: DNA-purification free PCR.
PCR 扩增基因组 DNA 片段是植物分子生物学方法（如基因分型）所必需的。虽然这是现代实验室中的常规分子技术，但在分析大量样本或在野外采集和储存样本时，仍然存在重大障碍。由于植物组织中存在各种抑制剂，直接从植物组织中进行 PCR 扩增通常无法成功，因此通常需要进行基因组 DNA 纯化，这涉及到繁琐且耗时的步骤或昂贵的材料，尤其是在使用商业试剂盒时。这些因素破坏了其可扩展性和在设备条件较差的环境中的应用。此外，植物组织和纯化的 DNA需要在适当的条件下储存，以避免降解。在这里，我们描述了一种低成本、高通量的 PCR 方法，可从植物组织中扩增基因组 DNA 片段，而无需进行 DNA 纯化或特殊的样品储存设备。在该方案中，取一小片植物组织将其研磨至纤维素基滤纸，直接将其放入含有吐温-20（一种非离子型清洁剂）的 PCR 混合物中，然后直接进行 PCR。我们还描述了制备自制 DNA 储存卡的步骤，该方法易于制作，可以与植物组织一起在室温下长时间储存，而无需任何特殊设备，使我们可以多次测试相同的样品。我们已经在至少 11 种植物物种中使用了这种方法，包括拟南芥、番茄、大豆、土豆、棉花和水稻。总之，我们的方法减少了劳动力和成本，从而提高了通量，使基于植物 DNA 的分子诊断检测方法在资源有限的环境（包括教室）中变得更加普及，并促进了野外样本的采集。 © 2021 Wiley Periodicals LLC. 基本方案 1：制作自制纤维素基 DNA 储存卡 基本方案 2：在 DNA 储存卡上研磨植物组织 基本方案 3：无需 DNA 纯化的 PCR
