难提取细菌基因组DNA提取方法的比较与优化  

Comparison and Optimization of Methods for Extraction of Problematic Bacteria Genomic DNA

作　　者：程实[1,2] 刘文丽[2] 舒鹏[1,2] 米志强[2] 安小平[2] 童贻刚[1,2] 

机构地区：[1]安徽医科大学,安徽合肥230032 [2]军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071

出　　处：《生物技术通讯》2016年第3期412-415,共4页Letters in Biotechnology

基　　金："艾滋病和病毒性肝炎等重大传染病防治"科技重大专项"十二五"实施计划(2013ZX10004-605;2013ZX10004-217;2013ZX10004-607;2011ZX10004-001);国家高技术研究发展计划(2014AA021402;2012AA022-003;2015AA020108);国家自然科学基金(81572045)

摘　　要：目的:比较并优化2种试剂盒提取以巨型球菌为代表的难提取细菌基因组DNA的方法,以获得高质量的DNA用于高通量测序,探索高通量测序样品的前期处理方法。方法:用Life Technologies公司的Charge Switchg DNA Mini Bacteria Kit和Roche公司的High Pure PCR Template Preparation Kit提取制备困难的巨型球菌基因组DNA,然后通过添加溶葡球菌酶和增加溶菌酶、RNase A、Protein K的投入量及延长消化时间优化2个试剂盒,对4种方法提取的细菌基因组DNA进行浓度、纯度和基因组完整性测定,选取质量良好的基因组进行高通量测序。结果:改良后的2个试剂盒提取的巨型球菌DNA浓度较高,分别达到78.4和67.8 ng/μL,可满足高通量测序的需求,并获得基因组全序列。结论:改良后的2种方法是提取制备困难的巨型球菌基因组DNA更好的方法。Objective：In order to obtain high-quality bacterial genomic DNA for next-generation sequencing （NGS） and explore the methods of sample processing,we compared and optimized the efficiency of two commercial DNA extraction kits for the extraction of problematic bacteria genomic DNA with representation of Macrococcus.Methods：We used two commercial DNA extraction kits,ChargeSwitch gDNA Mini Bacteria Kit of Life Technologies and High Pure PCR Template Preparation Kit of Roche,to extract problematic Macrococcus genomic DNA.Afterwards,the two kits were optimized by adding lysostaphin and improving amount of lysozyme,RNase A and Protein K,moreover,the time of enzyme digestion was also increased.Then we assessed these four extraction methods by using the parameters of concentrations,absorbance ratio and the integrity of obtained DNA.After that,genomic DNA with good quality were chosen for NGS.Results：Among all the four methods,the optimized ones obtained the highest concentration of Macrococcus with the mean concentration of 78.4 and 67.8 ng/μL respectively,meeting the requirements of NGS.We also assembled the complete genome sequences of Macrococcus.Conclusion：Our optimization provides a better choice of DNA extraction methods for problematic Macrococcus genomic DNA.

关 键 词：高通量测序 细菌基因组 DNA提取方法 

分 类 号：Q78[生物学—分子生物学]