Ahmed I, Islam M, Arshad W, Mannan A, Ahmad W, Mirza B
Allan Wilson Centre for Molecular Ecology and Evolution, Massey University, Palmerston North, New Zealand.
J Appl Genet. 2009;50(2):105-7. doi: 10.1007/BF03195661.
Polymerase chain reaction has found wide applications in modern research involving transformations and other genomic studies. For reproducible PCR results, however, the quantity and quality of template DNA is of considerable importance. A simple and efficient plant DNA extraction procedure for isolation of high-quality DNA from plant tissues is presented here. It requires maceration of plant tissue of about 1.0 cm(2) (e.g. of a leaf blade) in DNA extraction buffer (100 mM Tris-HCl, 100 mM EDTA, 250 mM NaCl) using 1.5-mL microfuge tubes, followed by cell lysis with 20% SDS, and DNA extraction with phenol: chloroform: iso-amyl alcohol (25:24:1). Hydrated ether is then used to remove polysaccharides and other contaminants from the DNA preparation. Average DNA yield is 20-30 microg cm(-2) for fresh tissues, and ratio of absorbance at 260 nm to absorbance at 280 nm is 1.5-1.8. The DNA is quite suitable for PCR using microsatellites, RAPD and specific markers for recombinant selection. Amplifications have been obtained for these markers by using template DNA extracted from fresh as well as frozen leaf tissues of various plants, including barley, oat, potato and tomato. DNA stored for more than 2 years has been successfully amplified with microsatellite markers, which shows suitability of this method after long-term storage of DNA. Besides, the ease of use and cost-effectiveness make the procedure attractive.
聚合酶链反应在涉及转化和其他基因组研究的现代研究中得到了广泛应用。然而，为了获得可重复的PCR结果，模板DNA的数量和质量至关重要。本文介绍了一种简单高效的从植物组织中提取高质量DNA的方法。该方法需要在1.5毫升微量离心管中，将约1.0平方厘米的植物组织（如叶片）在DNA提取缓冲液（100 mM Tris-HCl、100 mM EDTA、250 mM NaCl）中研磨，随后用20% SDS进行细胞裂解，并用苯酚：氯仿：异戊醇（25:24:1）提取DNA。然后使用水合乙醚去除DNA制剂中的多糖和其他污染物。新鲜组织的平均DNA产量为20 - 30微克/平方厘米，260纳米处的吸光度与280纳米处的吸光度之比为1.5 - 1.8。该DNA非常适合用于使用微卫星、RAPD和用于重组选择的特异性标记进行PCR。使用从包括大麦、燕麦、马铃薯和番茄在内的各种植物的新鲜和冷冻叶片组织中提取的模板DNA，已成功扩增出这些标记。用微卫星标记成功扩增了储存超过2年的DNA，这表明该方法在DNA长期储存后仍适用。此外，该方法的易用性和成本效益使其颇具吸引力。
