一种适合MSAP分析谷物干种子的DNA提取方法

An Efficient DNA Extraction Method for MSAP Analysis on the Dry Cereal Seeds

甲基化敏感扩增多态性(MSAP)是DNA甲基化的主要研究技术,获得高质量的DNA是MSAP分析的基础。针对玉米Zea mays(L.)等干种子富含多糖、蛋白质且与核酸结合紧密等特点,采用改良的CTAB-SDS法对其基因组总DNA进行提取,对DNA进行质量检测,使用限制性内切酶Eo RI和Hpa II酶切,进行MSAP分析。结果表明,改良CTAB法可从玉米等干种子中获得纯度高、完整性好的总DNA,其OD260 nm/OD280 nm处于1.86,OD260nm/OD230大于2.00,产率达1.8μg/mg,多糖、蛋白质和RNA等杂质被去除干净,无降解现象,所提取的DNA可被等限制性内切酶完全双酶切,进行MSAP分析得到大量清晰稳定的多态性条带。结果表明改进的CTAB-SDS法更适合于干玉米种子高质量总DNA的提取。

Methylation sensitive amplification polymorphism （MSAP） is the main technology of DNA methylation and to obtain high quality DNA is the basis of the MSAP analysis. The dry embyos of Zea may （L.） seeds are rich in polysaccharides, protein and closely bind to nucleic acid preventing DNA extraction. The genomic DNA of maize seed was extracted by improved CTAB-SDS method and DNA quantity was checked. DNA samples were digested by restriction enzymes EcoR I and Hpa II and MSAP analysis. The results showed that the genomic DNA was pure and integral without polysaccharides, protein, RNA and degradation. A260/A280 of the obtained DNA solution was 1.86 and A260/A230 ratios was greater than 2.00. The production rate was 1.8μg/mg. The total DNA can be digested completely and MSAP fragments amplified with DNA template showed clear hands, and good polymorphic, stability and repetition. The results showed that the improved CTAB-SDS method was more suitable for high quality DNA extraction from dry corn seed.