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Process for extracting microbe genome DNA in complex solid-state environment

An extraction method and microbial technology, applied in the direction of recombinant DNA technology, DNA preparation, etc., can solve the problems of DNA extraction obstacles, affecting molecular biology experiments, etc., and achieve the effect of low experiment cost

Inactive Publication Date: 2008-12-31
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this complex solid-state environment contains a large number of impurities, such as various metal ions and other inorganic substances, as well as various organic substances such as humus, which has caused considerable obstacles to the extraction of DNA and affected subsequent molecular biology. learning experiment

Method used

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  • Process for extracting microbe genome DNA in complex solid-state environment
  • Process for extracting microbe genome DNA in complex solid-state environment
  • Process for extracting microbe genome DNA in complex solid-state environment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Embodiment 1: Using the method of the present invention to extract the microbial genome DNA in the tidal flat mud of the Yangtze River Estuary in Chongming Island, Shanghai

[0017] The tidal flat is the tidal flooding zone between the high tide level and the low tide level of the coastal spring tide, and the tidal flat at the Yangtze Estuary of Chongming Island is not only infiltrated by sea water, but also washed by the Yangtze River. Therefore, a large amount of pollutants from the upper reaches of the Yangtze River have accumulated in the tidal flats, which also contain inorganic salts. In this saline-alkali environment rich in pollutants, most plants are difficult to grow, and only a few plant species exist near land. The microbial genome DNA in mudflat mud can be successfully extracted using aspects of the present invention.

[0018] The extraction process of the inventive method:

[0019] Weigh 0.84g of the sample, and extract it according to the following step...

Embodiment 2

[0022] Embodiment 2: Use the method described in the present invention and general method to the comparison of extracting microbial genome DNA from biogas slurry sample

[0023] Biogas digesters mainly use agricultural waste products, such as straw and other waste biomass materials, garbage and organic waste, and human and animal feces, and mix them to produce a mixed gas whose main component is methane under anaerobic conditions. Therefore, the composition of biogas slurry is quite complex, containing calcium, manganese, potassium, iron, copper, zinc and other metal ions, as well as humic acid, monosaccharides, polysaccharides, unsaturated fatty acids and certain antibiotics, which are harmful to DNA. The extraction and follow-up PCR and other molecular biology experiments caused great difficulties. Using the method described in the present invention to extract microbial genome DNA from biogas slurry samples is of higher quality than the DNA extracted using common methods.

...

Embodiment 3

[0031] Embodiment 3: Carry out PCR amplification 16S rRNA gene to DNA sample (using the method of the present invention and general method to extract microbial genome DNA from biogas slurry sample), the result shows that the DNA sample that uses the present invention to extract can carry out PCR smoothly Amplification, while those obtained by general methods cannot be amplified by PCR.

[0032] The DNA sample extracted in Example 2 was taken for PCR amplification. The PCR reaction system is 50μl: 10×buffer: 5μl, 10mM dNTP: 1μl, 10mM primer P0: 2.5μl, 10mM primer P6: 2.5μl, Taq enzyme (2U / μl): 1μl, DNA: 0.5μl, ddH2O: 37.5 μl. The sequence of the primers is: P0, 5'-GAGAGTTTGATCCTGGCTCAG-3', P6: 5'-CTACGGCTACCTTGTTACGA-3' (Weisburg et al., 1991), and the amplified target fragment is the full length of the 16S rRNA gene, about 1.5kb. The PCR parameters were designed as follows: initial denaturation for 5 minutes, 95°C; 30 cycles (denaturation for 30 seconds, 95°C; annealing for ...

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Abstract

The invention provides a method for directly extracting microbial genome DNA in the complex solid environment. The method comprises specific steps of: firstly adding an Extraction Buffer in a sample, then crushing bacteria, next using CTAB to remove protein and organic macromolecular substances under high ionic concentration, and finally extracting DNA from a solution by the conventional method; and the method is fast and convenient, and the acquired DNA can be used for operating the follow-up PCR equimolecular experiment.

Description

technical field [0001] The invention relates to a method for extracting microbial genome DNA. In particular, a method for extracting microbial genomic DNA in a complex solid-state environment. Background technique [0002] In the past two decades, scientists have gradually used molecular biology techniques to study microbial communities. This method can be relatively convenient to study functional populations in microbial communities, because it is very difficult to isolate and cultivate these microorganisms under existing laboratory conditions. of. Molecular biology techniques used in such research generally include PCR, DGGE (Denaturing Gradient Gel Electrophoresis), sequencing, oligonucleotide probes and FISH (Fluorescence In Situ Hybridization), etc. The 16S rRNA gene is the most effective and common molecular marker used in this type of research. Advances in these molecular biology methods enable us to conduct certain studies on uncultured microorganisms, and help us...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 宋任涛濮佳张荫雷贺其其袁轶伟马中良许政暟
Owner SHANGHAI UNIV
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