重金属污染土壤总DNA提取方法的研究——土壤预处理和硫酸铵铝在DNA提取中的应用

Application of soil pretreatments and AlNH_4(SO_4)_2 in DNA extraction from soils contaminated with heavy metals

【目的】从土壤中获得高纯度、高得率和完整性好的总DNA,为重金属污染土壤微生物群落多样性的分析奠定基础。【方法】将一定浓度的硫酸铵铝增补入DNA提取液中,分别联合不同方式的土壤预处理,对所提取的土壤总DNA进行完整性、纯度和得率分析。【结果】TENP-AlNH4(SO4)2法、ABG-AlNH4(SO4)2法、Wash-AlNH4(SO4)2法和试剂盒4种提取方法获得的DNA片段均在23 kb左右,总DNA完整;Wash-AlNH4(SO4)2法提取的DNA纯度较高,A260/A230达到2.00,A260/A280达到1.62;ABG-AlNH4(SO4)2法的DNA得率最高,达到1 010μg/g土壤;这两种方法提取的DNA在纯度和浓度上均达到后续PCR等分子实验要求。通过扩增提取的土壤总DNA中16S rRNA基因,表明合适浓度的硫酸铵铝能有效去除土壤中的PCR干扰因子。【结论】本研究将土壤的预处理和一定浓度的硫酸铵铝联合使用,获得理想的重金属污染土壤的总DNA。

[Objective] The aim of this study was to obtain DNA with high purity, high yield and good integrity from the soil, for the analysis of the microbial diversity of heavy metal contaminated soil. [Methods] Accompanied by different soil pretreatments, AlNH4（SO4）2 was added in the DNA extraction buffer to analyze the yield, purity and integrity of the total DNA extracted from the soil. [Results] The DNA fragments extracted from the four different methods, TENP-AlNH4（SO4）2, ABG-AlNH4（SO4）2, Wash-AlNH4（SO4）2 and Reagent Kit, were all intact, and the sizes of them were all about 23 kb. The Wash-AlNH4（SO4）2 method gave the purest DNA with 2.13 at A260/A230 and 1.62 at A260/A280, while the yield from ABG-AlNH4（SO4）2 method was the highest with 1 010 μg DNA per gram soil. Both methods could provide DNA with sufficient purity and concentration for the following molecular experiments such as PCR. Through the amplification of 16S rRNA gene from the extracted soil DNA, PCR inhibitors in the soil could be efficiently removed by AlNH4（SO4）2 with appropriate concentrations. [Conclusion] By combining soil pretreatment and utilization of AlNH4（SO4）2, we obtained the desired total DNA from the soil contaminated with heavy metals.