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Method for separating metagenome deoxyribonucleic acid (DNA) and total ribonucleic acid (RNA) of microorganism

A metagenomic and microbial technology, applied in the direction of recombinant DNA technology, DNA preparation, etc., can solve the problems of insufficient amount of nucleic acid required for analysis, deviation of analysis results, and low recovery rate, etc., to achieve unlimited capacity, good quality, and high recovery rate Effect

Inactive Publication Date: 2014-03-19
CENT SOUTH UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The cost of separation using kits is high, and the recovery rate will decrease when the processing volume exceeds the capacity of the adsorption column, resulting in serious losses; when using enzyme treatment, although RNA in DNA or DNA in RNA can be effectively removed, However, the waste is serious, especially for some difficult-to-sample or limited-sample samples with a small amount of extracted nucleic acid, which may lead to insufficient amount of nucleic acid required for analysis and need to be amplified, and amplification often leads to deviations in analysis results or distortion

Method used

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  • Method for separating metagenome deoxyribonucleic acid (DNA) and total ribonucleic acid (RNA) of microorganism
  • Method for separating metagenome deoxyribonucleic acid (DNA) and total ribonucleic acid (RNA) of microorganism
  • Method for separating metagenome deoxyribonucleic acid (DNA) and total ribonucleic acid (RNA) of microorganism

Examples

Experimental program
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Effect test

Embodiment 1

[0029]In this example, the cell lysate used is a strain sample, and the strains used are all from the pure bacteria preserved in the strain bank of the Key Laboratory of Biometallurgy, Ministry of Education, School of Resource Biology, Central South University. Water (pH1-3 or so), can grow at a lower pH, has the ability to oxidize ferrous and sulfur, and is generally used in microbial hydrometallurgical production; among them, Acidithiobacillus.ferrooxidans (A.f) is acidophilic ferrous oxide Thiobacillus, the optimum growth temperature is 25-30°C, the optimum pH is 1.5-2, and obtains energy from ferrous oxide ions; The optimum pH is 2-2.5, using reduced sulfide and elemental sulfur as energy substances; At.albertlensis (A.t) is mesophilic acidophilus Thiobacillus acidophilus, the optimum growth temperature is 30°C, the optimum pH is 1.8, using elemental sulfur and reduced form Sulfide growth; Leptospirillum.ferriphilium (L.f) is an iron-loving Leptospira, with an optimum grow...

Embodiment 2

[0046] In this embodiment, the cell lysate used is environmental samples, such as water samples and mud samples in mining areas. Its specific steps are consistent with those in Example 1.

[0047] image 3 3a is M, using lithium chloride to separate the DNA of copper mine water samples I, II, gold mine water samples and mud samples; 3b is M, using lithium chloride to separate copper mine water samples I, II, gold mine water samples and It can be seen from the figure that the RNA of the mud sample can be successfully separated into large fragments of DNA and small fragments of RNA by using this method to successfully separate nucleic acids (including DNA and RNA) simultaneously extracted from environmental samples.

[0048] The test results can be compared with the examples as follows.

[0049] Figure 6 The 6a bands in the middle are M, blank control, A.f(1), A.c(2), A.t(3) and copper mine water samples II(4), I(5) and gold mine water samples (6) and mud samples (7) Revers...

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Abstract

The invention relates to the technical field of separation of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and discloses a method for separating metagenome DNA and total RNA of a microorganism. The method comprises the following steps: S1, extracting total ribonucleic acid, namely preparing cell lysis buffer, removing protein and lipids from the cell lysis buffer by using an organic solvent; S2, separating total RNA, namely adding a lithium chloride solution to the total ribonucleic acid in the step S1, separating out the total RNA by adopting a centrifuging method after standing at a low temperature, and transferring and placing the metagenome DNA in supernate; S3, participating the metagenome DNA, namely taking out the supernate transferred and placed in the step S2, participating the metagenome DNA in the supernate by using pre-cooled isopropanol or ethanol, and separating out the metagenome DNA by adopting the centrifuging method after stewing at low temperature; S4, purifying the metagenome DNA and the total RNA, washing and purifying the total RNA and the metagenome DNA obtained in the steps S2 and S3 by adopting ethanol, and obtaining the purified total RNA and metagenome DNA by adopting the centrifuging method. The method has the advantages of being good in separation effect, high in recovery rate, low in cost, and simple in operation.

Description

technical field [0001] The invention relates to the technical field of DNA and RNA separation, and more specifically relates to a method for separating microbial metagenomic DNA and total RNA. Background technique [0002] At present, there are many existing reports on the method for nucleic acid extraction simultaneously, including animal tissue cells without cell walls (Deborah A. Triant, Andrew Whitehead. Simultaneous Extraction of High-Quality RNA and DNA from Small Tissue Samples [J]. Journal of Heredity, 2009, 100(2): 246-250), plant tissues with tough cell walls (Liu Shenghong, Liu Wen, Liu Mingfeng, etc. A method for efficiently extracting kiwifruit DNA and RNA [J]. Biotechnology Bulletin, 2011 , 9:171-175), medical materials (M.Bauer,D.Patzelt.A method for simultaneous RNA and DNA isolation from dried blood and semen stains[J].Forensic Science International[J].2003,136(1- 3): 76-78.) or other environmental samples (Carla M.Zammita, Lesley A.Mutcha, v, et al. The re...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 刘新星谢建平云慧邱冠周
Owner CENT SOUTH UNIV
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