煤地质环境微生物总基因组DNA提取方法的优化

doi:10.13560/j.cnki.biotech.bull.1985.2017-1145

生物技术通报 ›› 2018, Vol. 34 ›› Issue (9): 177-183.doi: 10.13560/j.cnki.biotech.bull.1985.2017-1145

煤地质环境微生物总基因组DNA提取方法的优化

杨秀清¹, 陈彦梅¹, 吴瑞薇¹, 王保玉^2,3, 韩作颖^2,3

1. 山西大学生物技术研究所化学生物学与分子工程教育部重点实验室,太原 030006;
2. 煤与煤层气共采国家重点实验室,晋城 048000;
3. 易安蓝焰煤与煤层气共采技术有限责任公司,晋城 048000

收稿日期:2017-12-29 出版日期:2018-09-26 发布日期:2018-10-10
作者简介:杨秀清,男,副教授;研究方向：生物转化与生物催化;E-mail：xiuqyang@sxu.edu.cn
基金资助:
山西自然科学与煤层气联合基金会项目（2015012002）,山西省重点科技攻关项目（MQ2014-03）

Optimization of Extracting Method for Microbial Genome DNA in Coal Geological Environment

YANG Xiu-qing¹, CHEN Yan-mei¹, WU Rui-wei¹, WANG Bao-yu^2,3, HAN Zuo-ying^2,3

1. Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education,Institute of Biotechnology,Shanxi University,Taiyuan 030006;
2.State Key Laboratory of Coal and CBM Co-mining,Jincheng 048000;
3.Yi’an Lanyan Coal and Coal-bed Methane Simultaneous Extraction Technology Co.,Ltd.,Jincheng 048000;

Received:2017-12-29 Published:2018-09-26 Online:2018-10-10

摘要/Abstract

摘要： 高质量的DNA是进行分子生物学研究的基础。通过对传统DNA抽提方法（酚-氯仿法）进行改进,并以Rhodococcus sp.R04和煤粉为实验材料对细胞破碎条件进行优化,建立了一种可用于煤地质环境微生物基因组DNA高效提取的改良方法。以改良法和商业试剂盒提取煤地质环境微生物基因组DNA,通过琼脂糖凝胶电泳、细菌及古菌特异性片段的PCR扩增来评价所提取DNA的质量。改良法和试剂盒法均能获得煤地质环境微生物基因组DNA,并能用于多种特异性PCR扩增。与试剂盒提取的DNA相比,改良法获得的DNA片段主带明显,约占总DNA含量的50%,分子量大小接近23 kb,并且提取量大,约为试剂盒的5-10倍。同时,能用于如DNA文库构建和宏基因组测序等。此外,改良法所用试剂普通,价格便宜,提取的煤地质环境微生物基因组DNA质量较高,适于实验室和科学研究。

关键词: 煤地质环境微生物, 基因组DNA提取, DNA质量, PCR扩增

Abstract: High-quality DNA is the basis for molecular biology research. By improving the traditional method of extracting DNA（phenol-chloroform method）and optimizing the cell disruption conditions while having Rhodococcus sp.R04 and pulverized coal as experimental materials,a modified method of efficiently extracting genomic DNA from microbes in coal geological environment has been established. The microbial genomic DNAs extracted by this modified method and the commercial kit were evaluated by agarose gel electrophoresis and PCR amplifications of bacterial and archaea specific fragments. The results showed that the microbial genome DNAs from coal geological environment were obtained by both the modified method and the kit method,and used as temple for various specific PCR amplifications. Compared with the extracted DNA with the commercial kit,the DNA by the modified method had obvious main band,accounted about 50% of total DNA content,and the molecular weight of the DNA was nearly 23 kb. Also,the extraction amount of DNA was very large,and was about 5-10 times that by the commercial kit. Moreover,DNA by the modified method can be used in many purposes such as DNA library construction and metagenomic sequencing. Consequently,the modified method for extracting microbial genomic DNA from coal geological environment is not only cheap and effective,also the extracted DNA is in high-quality,thus it is very suitable for laboratory and scientific researches.

Key words: microorganism in coal geological environment, genomic DNA extraction, DNA quality, PCR amplification

杨秀清, 陈彦梅, 吴瑞薇, 王保玉, 韩作颖. 煤地质环境微生物总基因组DNA提取方法的优化[J]. 生物技术通报, 2018, 34(9): 177-183.

YANG Xiu-qing, CHEN Yan-mei, WU Rui-wei, WANG Bao-yu, HAN Zuo-ying. Optimization of Extracting Method for Microbial Genome DNA in Coal Geological Environment[J]. Biotechnology Bulletin, 2018, 34(9): 177-183.

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煤地质环境微生物总基因组DNA提取方法的优化

Optimization of Extracting Method for Microbial Genome DNA in Coal Geological Environment

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