改良CTAB法提取新疆一枝蒿干叶基因组DNA

Extraction of Genomic DNA from Dry Leaves of Artemisia rupestris by Modified CTAB Methods

目的：从自然干燥的新疆一枝蒿中提取高质量的基因组DNA,为该药材的分子生物学研究提供参考。方法：采用2种改良十六烷基三甲基溴化铵（CTAB）法与常规CTAB法提取新疆一枝蒿中基因组DNA,通过加适量聚乙烯吡咯烷酮（PVP）研磨样本,在核裂解前用细胞核裂解液（STE）洗涤多酚类和多糖类物质,利用95%乙醇室温沉淀DNA等措施。改良CTAB法2与1相比不用液氮研磨样本。通过琼脂糖凝胶电泳、紫外分光光度法和相关序列扩增多态性（SRAP）检测提取DNA的质量。结果：2种改良CTAB法所得DNA质量均优于常规CTAB法,DNA完整性较好,A260 nm/A280 nm在1.8~2.0,多糖类杂质去除较为干净。SRAP检验条带清晰、多态性良好。结论：2种改良CTAB法均适用于高质量新疆一枝蒿干叶基因组DNA的提取,得到的DNA纯度和完整性均较理想,可为后续分子生物学研究提供良好模版。

Objective： To extract high quality genomic DNA from dry leaves of Artemisia rupestris dried naturally and lie a foundation for further molecular experiments. Method： Three different methods, conventional hexadecyl trimethyl ammonium bromide （CTAB） method and modified CTAB methods were applied to isolate genomic DNA from dry leaves of A. rupestris. Two modified CTAB methods were different from conventional CTAB method in three ways, nucleus lysate （STE） was used for removal of impurities, polyvinyl pyrrolidone （PVP） was added when grinding samples and 95% ethanol for precipition of DNA in room temperature. These two modified CTAB methods were different from whether liquid nitrogen was added while grinding samples. Purity and integrality of DNA were detected by UV spectrophotometry, agarose gel electrophoresis and sequence-related amplified polymorphism （SRAP）. Result： Higher purity DNA was obtained by improved CTAB methods by comapred with conventional CTAB method, value of A260nm/A280 nm was between 1.8-2.0, polysaccharides was removed relatively clear. SRAP produced clear polymorphic patterns. Conclusion： These two modified CTAB methods are suitable for DNA extraction from dry leaves of A. rupestris, purity and integrality of isolated DNA can be used in further molecular biology research.