Kang Tae-Jin, Yang Moon-Sik
Institute of Basic Science, Chonbuk National University, Jeonju 561-756, South Korea.
BMC Biotechnol. 2004 Sep 2;4:20. doi: 10.1186/1472-6750-4-20.
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.
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.
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.
从愈伤组织和植物中提取用于PCR的高质量DNA的方法效率不高，因为这些方法需要将组织在液氮中研磨，然后用乙醇沉淀DNA沉淀，洗涤并干燥沉淀等。迫切需要一种快速简单的方法，尤其是在需要分析数百个样品时。在此，我们描述了一种从愈伤组织、各种野生型和转基因植物中分离高质量基因组DNA用于PCR扩增和酶切的简单有效方法。
我们开发了一种新的快速可靠的基因组DNA提取方法。使用我们开发的方法，植物基因组DNA提取可在30分钟内完成。方法如下。使用手动匀浆器将植物组织与盐DNA提取缓冲液匀浆，并用苯酚：氯仿：异戊醇（25：24：1）提取。离心后，上清液直接用作PCR的DNA模板，成功扩增了来自各种植物来源的RAPD和转基因植物的特定外源基因。沉淀上清液后，DNA被限制性内切酶完全消化。
这种DNA提取方法在时间和所需植物样品量方面都保证了简单、快速和高效。此外，这种方法不需要昂贵的设备来提取植物基因组DNA。
