Method and kit for extracting total DNA of soil and sediment

An extraction method and sediment technology, applied in the field of soil and sediment total DNA extraction methods and extraction kits, can solve the problems of high toxicity, high pollution, pollution, long time, etc., and achieve broad application prospects and high sample purity.

Inactive Publication Date: 2011-10-05
OCEAN UNIV OF CHINA
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Problems solved by technology

[0003] At present, there is still no agreed-upon better method for obtaining total DNA from soil and sediments, and the commonly used enzymatic cleavage methods and chemical extraction methods can only work on sensitive bacteria in samples, and cannot remove samples. humic acid in
These methods have artificially changed the real microbial community structure in the environment, and have the following problems: 1) The DNA extraction process is complicated, takes too long, and requires more large-scale instruments and equipment, such as the enzyme extraction method that requires a long water bath process ; 2) The general DNA extraction process often uses highly toxic and highly polluting chemical reagents, such as phenol / chloroform and other "three-inducing" reagents in the DNA purification process; 3) The quality of the extracted DNA is poor, there are many fragments, and there are humic acids , RNA, protein and other pollution; 4) The amount of DNA is too small, especially to obtain DNA from sediments, which cannot meet the needs of downstream experiments

Method used

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  • Method and kit for extracting total DNA of soil and sediment
  • Method and kit for extracting total DNA of soil and sediment
  • Method and kit for extracting total DNA of soil and sediment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1: the selection of humic acid complexing agent concentration

[0063] This kit was used to investigate the effect of the concentration of humic acid complexing agent on the extraction efficiency by taking wetland soil, marine sediment and farmland soil as objects. The sample size is 0.3g, wherein the concentration of calcium chloride solution is 0, 0.1, 0.2, 0.3, 0.4, 0.5mol / L, respectively, 1ml is added, and the extraction is carried out according to the method of the present invention, and the time spent is about 60min. The extracted DNA was electrophoresed, stained, photographed, and qualitatively and quantitatively analyzed using an ultraviolet spectrophotometer. The results show that when the concentration of calcium chloride solution is 0 and 0.1mol / L respectively (i.e. the ratio of sample wet weight and calcium ion ≥ 3g / mmol), although the recovery rate of DNA is higher, it is 10~15μg / g dry soil, However, the extracted DNA solution is yellow and cont...

Embodiment 2

[0064] Example 2: Bacterial Cell Lysis Vortex Oscillation Time Selection

[0065] Using this kit, the vortex oscillation time after adding cell lysate and denaturant was optimized for 0.3g of wetland soil and farmland soil respectively. Wherein the humic acid complexing agent uses 1ml of 0.2mol / L calcium chloride solution, pH 7.5, and the vortex oscillation time during cell lysis is selected 5min, 10min, 15min respectively, and is extracted according to the method of the present invention, and the time spent is about 70min. DNA electrophoresis, staining, photography, and qualitative and quantitative analysis using an ultraviolet spectrophotometer. The results showed that the DNA recovery rate of the environmental samples shaken for 5 minutes was significantly lower, lower than 1.0 μg / g dry soil; but after more than 15 minutes, the DNA recovery rate did not increase significantly, and a large number of DNA fragments appeared, which is necessary for long-term Molecular biology ...

Embodiment 3

[0066] Example 3: Extraction of total DNA from different soil samples

[0067] Using this kit and determined extraction conditions, the total DNA in farmland soil, grassland soil, wetland soil, and marine sediments was extracted respectively, and the extraction was carried out according to the method of the present invention, and it took about 60 minutes. Take 10 μL of DNA for electrophoresis, the gel is stained with EB, the gel imaging system is photographed, and the qualitative and quantitative analysis is carried out with a UV spectrophotometer. The electrophoresis results are as follows: figure 1 shown. According to the figure, the kit can obtain total DNA from different soils and sediments, the length of DNA fragments is 10-20kb, there is no degradation and no RNA pollution. The ultraviolet absorption detection was carried out on each sample respectively, indicating that the obtained DNA samples formed a uniform DNA absorption peak at 260nm, and there was no obvious prot...

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Abstract

The invention relates to a method and a kit for extracting total DNA of soil and sediment. The method comprises the following steps of: dissolving and removing humic acid from the soil and sediment to the greatest extent by using a humic acid dissolving solution; performing complex-precipitation on the residual humic acid; randomly cracking and releasing genomic DNA in bacteria; centrifuging to remove complexed humic acid and protein from the solution; and regulating supernate containing DNA by using an acid-base regulating agent and a high-concentration salt solution, and specifically adsorbing by using a silica gel adsorption film to make the genomic DNA purified and recovered. By the method, a plurality of samples can be extracted in short time, the normal operating time is less than 70 minutes, and massive DNA samples having high purity and suitable for downstream analysis can be obtained. By the kit established according to the method, the obtained DNA samples have high purity without degradation and can be directly used for subsequent molecular biology analysis; meanwhile, a step of purifying the DNA by toxic reagents such as phenol and chloroform in the traditional DNA extraction method is avoided, and the invention has wide application prospect.

Description

technical field [0001] The invention belongs to the technical field of separation and purification of soil microbial genome DNA, and in particular relates to a soil and sediment total DNA extraction method and an extraction kit. Background technique [0002] Molecular fingerprinting technology has become a commonly used microbial ecology technology in the fields of microbial ecology, environmental biology, environmental engineering, etc. However, the extraction technology of total DNA in environmental samples (such as soil, ocean and lake sediments, etc.) The key steps and core content of the system have received widespread attention. The specificity, concentration, and purity of total DNA extraction in soil and sediment directly affect the objective authenticity of the microbial community structure, which is one of the most controversial contents in the review process of foreign journals in recent years. [0003] At present, there is still no agreed-upon better method for ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 赵阳国李新伟白洁田伟君王俊彩刘茹
Owner OCEAN UNIV OF CHINA
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