Yu Guotai, Hatta Asyraf, Periyannan Sambasivam, Lagudah Evans, Wulff Brande B H
John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK.
Department of Agriculture Technology, Universiti Putra Malaysia, Serdang, Malaysia.
Methods Mol Biol. 2017;1659:207-213. doi: 10.1007/978-1-4939-7249-4_18.
DNA is widely used in plant genetic and molecular biology studies. In this chapter, we describe how to extract DNA from wheat tissues. The tissue samples are ground to disrupt the cell wall. Then cetyltrimethylammonium bromide (CTAB) or sodium dodecyl sulfate (SDS) is used to disrupt the cell and nuclear membranes to release the DNA into solution. A reducing agent, β-mercaptoethanol, is added to break the disulfide bonds between the cysteine residues and to help remove the tanins and polyphenols. A high concentration of salt is employed to remove polysaccharides. Ethylenediaminetetraacetic acid (EDTA) stops DNase activity by chelating the magnesium ions. The nucleic acid solution is extracted with chloroform-isoamyl alcohol (24:1) or 6 M ammonium acetate. The DNA in aqueous phase is precipated with ethanol or isopropanol, which makes DNA less hydrophilic in the presence of sodium ions (Na+).
DNA在植物遗传和分子生物学研究中被广泛应用。在本章中，我们将描述如何从小麦组织中提取DNA。将组织样品研磨以破坏细胞壁。然后使用十六烷基三甲基溴化铵（CTAB）或十二烷基硫酸钠（SDS）破坏细胞膜和核膜，使DNA释放到溶液中。添加还原剂β-巯基乙醇以断裂半胱氨酸残基之间的二硫键，并有助于去除单宁和多酚。采用高浓度盐去除多糖。乙二胺四乙酸（EDTA）通过螯合镁离子来抑制DNase活性。用氯仿-异戊醇（24:1）或6M醋酸铵提取核酸溶液。水相中的DNA用乙醇或异丙醇沉淀，这使得DNA在钠离子（Na+）存在下亲水性降低。
