Rapid and reliable extraction of genomic DNA from various wild-type and transgenic plants
Rapid and reliable extraction of genomic DNA from various wild-type and transgenic plants
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DOI：
10.1186/1472-6750-4-20
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发表时间：
2004-09-02
影响因子：
3.5
通讯作者：
Yang, MS
中科院分区：
文献类型：
--
作者：
Kang, TJ;Yang, MS
背景资料：从愈伤组织和植物中提取PCR质量DNA的DNA提取方法不是时间效率高的，因为它们需要在液氮中研磨组织，然后在乙醇中沉淀DNA颗粒，洗涤和干燥颗粒等。迫切需要快速简单的程序，特别是当需要分析数百个样品时。在这里，我们描述了一种简单有效的方法，从愈伤组织，各种野生型和转基因plants.Results分离高质量的基因组DNA的PCR扩增和酶切。利用我们开发的方法，植物基因组DNA的提取可以在30分钟内完成。植物组织用盐DNA提取缓冲液使用手动匀浆器匀浆，并用苯酚：氯仿：异戊醇（25：24：1）提取。离心后的上清液直接作为PCR的DNA模板，成功地扩增出了不同来源植物的RAPD和转基因植物的特异性外源基因。沉淀后的上清液中，DNA完全消化的限制性内切酶。结论：这种DNA提取程序承诺简单，快速，高效，无论是在时间和所需的植物样品的量。此外，该方法不需要用于植物基因组DNA提取的昂贵设施。
Background: DNA extraction methods for PCR-quality DNA from calluses and plants are not time efficient, since they require that the tissues be ground in liquid nitrogen, followed by precipitation of the DNA pellet in ethanol, washing and drying the pellet, etc. The need for a rapid and simple procedure is urgent, especially when hundreds of samples need to be analyzed. Here, we describe a simple and efficient method of isolating high-quality genomic DNA for PCR amplification and enzyme digestion from calluses, various wild-type and transgenic plants.Results: We developed new rapid and reliable genomic DNA extraction method. With our developed method, plant genomic DNA extraction could be performed within 30 min. The method was as follows. Plant tissue was homogenized with salt DNA extraction buffer using hand-operated homogenizer and extracted by phenol: chloroform: isoamyl alcohol (25:24:1). After centrifugation, the supernatant was directly used for DNA template for PCR, resulting in successful amplification for RAPD from various sources of plants and specific foreign genes from transgenic plants. After precipitating the supernatant, the DNA was completely digested by restriction enzymes.Conclusion: This DNA extraction procedure promises simplicity, speed, and efficiency, both in terms of time and the amount of plant sample required. In addition, this method does not require expensive facilities for plant genomic DNA extraction.