Method for simultaneously extracting microbial genome deoxyribonucleic acid (DNA) and total ribonucleic acid (RNA) in mining area environmental sample

Abstract

The invention discloses a method for simultaneously extracting microbial genome deoxyribonucleic acid (DNA) and total ribonucleic acid (RNA) in a mining area environmental sample. The method comprises the following steps: S1, pretreatment of an environmental sample: collecting microorganisms in a liquid sample by a centrifuging method, or removing impurities out of a solid sample by a filtering method; S2, crushing cells: mixing the sample pretreated in the step S1 with quartz sand, adding liquid nitrogen, grinding for three times, adding PIPES extraction buffer of which the pH value is 7.0 and a sodium dodecyl sulfate solution, splitting cells at 65 DEG C for 1 hour; S3, purifying and participating nucleic acid: collecting supernate by the centrifuging method at the end of splitting of cells, adding an extraction agent to the supernate to centrifugally extract protein and lipids, participating the supernate by using isopropanol, and centrifuging so as to obtain the total ribonucleic acid; separating the total ribonucleic acid, so as to obtain the metagenome DNA and the total RNA. The method is low in cost and is capable of simultaneously extracting the metagenome DNA and the total RNA with high purity and good integrity from the mining area environmental sample.

Description

technical field [0001] The invention relates to the technical field of DNA and RNA separation, and more specifically relates to a method for simultaneously extracting microbial genome DNA and total RNA from environmental samples in mining areas. Background technique [0002] A major obstacle to the understanding, development and utilization of microbial resources in the environment is the difficulty in obtaining pure culture. According to reports, currently about 99% of environmental microorganisms have not been purely cultured due to unclear physical and chemical conditions required for growth (Pace, N.R.A molecular view of microbial diversity and the biosphere. Science. 276(5313), 1997: 734-740), Therefore, there are a large number of unknown microbial resources in the environment that need to be researched, developed and utilized urgently. One of the key technologies of biometallurgy is to obtain high-efficiency leaching strains from the mining area that can withstand ex...

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Problems solved by technology

Since these research methods are all based on the analysis of genomic DNA or mRNA, and the quality and quantity of nucleic acid will affect the reliability of data analysis (Demeke, T. & Jenkins, G.R. Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits.Anal Bioanal Chem.396(6), 2010:1977-1990), the traditional step-by-step extraction method of DNA and mRNA will cause "time difference", which makes it difficult to "synchronize" the analysis results, and, due to different Different nucleic acid extraction methods have been used in some studies, and different extraction methods will have an impact on the research results and cause differences (Inceoglu, O., Hoogwout, E.F., Hill, P. & van Elsas, J.D. Effect of DNA Extraction Method on the Apparent Microbial Diversity of Soil.Appl Environ Microb.76(10), 2010: 3378-3382), which makes it difficult to compare between different studies. Therefore, it is necessary to formulate a standard technique for the simultaneous extraction of genomic DNA and total RNA from samples in mining areas very necessary

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