YANG Xue-zhen,LIU Xiao-xue,SONG Jian,et al.Extraction of Soybean DNA by Improved High Salt and High pH Method[J].Soybean Science,2020,39(04):549-554.[doi:10.11861/j.issn.1000-9841.2020.04.0549]
改良高盐高pH法提取大豆DNA
- Title:
- Extraction of Soybean DNA by Improved High Salt and High pH Method
- Keywords:
- Soybean; DNA extraction; High salt and high pH; CTAB
- 文献标志码:
- A
- 摘要:
- 大豆除了含有大量的脂类和蛋白质外,还含有糖类和酚类物质,为解决难于从大豆中提取高质量DNA和传统提取方法耗时长的问题,本研究以中豆41与天隆一号的幼嫩叶片(V3)和成熟种子作为材料,对高盐低pH法进行改良,提高其裂解液pH值,同时结合冷冻裂解法缩短裂解时间,使用不同pH值醋酸钠纯化DNA,并与经典CTAB法进行比较,评价其提取DNA的效果及其应用范围。结果表明,高盐高pH法对成熟种子的DNA提取效率较高,且蛋白与RNA污染少。CTAB法提取的DNA虽然完整性较高,但是耗时较长,蛋白与RNA污染较多。另外,高盐高pH法提取的DNA PCR扩增条带清晰,与常规CTAB法提取DNA扩增效果一致,但其DNA质量不能满足高通量测序要求。综上,改良的高盐高pH法是一种快速有效的大豆DNA提取方法,可满足常规分子试验的要求。
- Abstract:
- Soybean contains substantial amount of protein and lipids, and sugar and phenols as well, which made the nucleic acid (DNA/RNA) extraction rather difficult and traditional DNA extraction methods are tedious and time-consuming. In the present study, high salinity and low pH methods was improved by elevating the pH value of lysis solution using mature seeds and leaves of Zhongdou 41 and Tianlong 1, respectively. The extraction process was shorten by the application of repeated freeze pyrolysis and DNA was purified by acetate sodium of distinct pH. The improved high salinity and high pH method was then evaluated in terms of effect, efficiency, and application scope in comparison with CTAB method. Results showed that the DNA extraction efficiency of high salinity and high pH method from seeds was significantly higher than CTAB with less contamination of protein and RNA residuals, but with less DNA integrity. Whereas, the CTAB yielded DNA with relatively higher DNA integrity but with more contamination and slower extraction speed. Besides, during the PCR amplification, the DNA extracted by high salinity and high pH showed clearly bands, which was similar with that by CTAB, however, which can not meet the standard of high throughput sequencing. The improved DNA extraction method was suitable for rapid DNA extraction in soybean, which could meet the demand of daily-use molecular experiments.
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