天山1号冰川冻土微生物总DNA提取方法的比较和优化

Comparison and Optimization of Methods for Metagenomic DNA Extraction from the Frozen Soil of the Glacier No.1 in the Tianshan Mountains

建立一种满足天山冰川冻土微生物宏基因组文库构建要求的DNA提取方法,为低温乳糖酶基因的筛选奠定基础。通过对DNA纯度与得率进行比较,从4种DNA提取方法中选择提取DNA得率和纯度最高的TEN法,进一步通过增加预处理、反复提取等步骤进行了改良和优化。SDS-CTAB法、TENP预处理法、TEN法和Zhou法4种提取方法中,TEN法的DNA得率为(392±44)ng/g冻土,A260/A280和A260/A230分别为(1.82±0.40)和(0.97±0.38),最接近理想指标。对TEN法进行优化改良发现,DNA得率最高达到(449±61)ng/g冻土,且A260/A280和A260/A230值分别为(1.92±0.15)和(2.03±0.31)。以16S rDNA和26S rDNA为引物的PCR扩增和酶切实验验证了所提取冻土宏基因组DNA的质量满足后续分子生物学的要求。改良优化过的TEN法是适合天山1号冰川冻土的一种经济有效的DNA提取方法,为构建冻土低温微生物宏基因组文库奠定了基础。

This study is aiming to establish a method for total DNA extraction from the frozen soil of the Glacier No.1 in the Tianshan Mountains.Four DNA extraction methods(SDS-CTAB,TENP,TEN and Zhou method)were used for DNA extraction from the frozen soil,and the yield,purity and integrity of the total DNA produced were compared.Among the four extraction methods tested,TEN method produced the highest purity with a A260/A280 and A260/A230 ratio of(1.82±0.40)and(0.97±0.38),respectively,and the yield of TEN method was(392±44)ng/g frozen soil.We further modified the TEN method by adding a pretreatment step.The A260/A280 and A260/A230 ratio of the extracted DNA reached(1.92±0.15)and(2.03±0.31),respectively,and the DNA yield of TEN4 method reached(449±61)ng/g frozen soil.16S rDNA,26S rDNA PCR analysis,and restriction digestion analysis of the DNA extracted by TEN method further proved that the DNA was qualified for the construction of a meta-genomic DNA library.In summary,a modified TEN method suitable for DNA extraction from the frozen soil of the Glacier No.1 in the Tianshan Mountains was established,which could be used for meta-genomic DNA library construction.