Comparison of results of extracting soil DNA from different brand kits

1 Introduction

Extracting high concentrations, large fragments, high diversity, and representative soil microbial total DNA is essential for soil microbial diversity research. However, soil is a very complicated heterogeneous system, which contains humic acid and humic acid analogues, phenolic compounds, heavy metal ions, etc., which cannot be effectively removed during DNA extraction, which will directly affect subsequent PCR amplification. Molecular manipulation such as nucleic acid hybridization, endonuclease digestion, and the like.

The study of complex microbial ecosystems using traditional microbiological methods must first be carried out for the isolation and culture of microorganisms. Due to the complexity of the soil microbial ecosystem and the limitations of culture conditions, the microorganisms that can be cultured in the laboratory are less than 1% of the total number of soil microorganisms . A considerable number of microorganisms are not recognized because they cannot be cultured ^[1] .

The extraction and purification of soil microbial DNA is a time-consuming and cumbersome process. Many scholars have been exploring faster, more efficient and low-cost methods for soil DNA extraction. Since the mid-1980s, microbiologists have begun to study soil microbes directly using culture-free molecular microbial ecology ^[2] , which must extract DNA from soil microbial communities. Volossiouk ^[3] used liquid nitrogen grinding and SDS (sodium dodecyl sulfate) to decompose microbial cells to extract soil DNA, and Tsai ^[4] used lysozyme and SDS to extract soil DNA, and the DNA extracted by these methods. More impurities, seriously affect the results of subsequent research ^[5-6] . Later, Bourrain et al ^[7] extracted the DNA of the microbial community from compost in activated sludge and LaMontagne et al. ^[8] . However, there is still no rapid, efficient, low cost and high quality method for soil DNA extraction. This article purchased the soil DNA kit commonly used in the market and compared the extraction effects.

2. Materials and methods

2.1 Materials

The soil samples used for DNA extraction were collected from the barren soil of the Huishan Central Park lawn in Huishan District, Wuxi City (1 to 10 cm topsoil; sample No. 1), the activated sludge on the pond side (sample No. 2), and the rot of the grove. Soil (1 to 10 cm topsoil; sample No. 3).

2.2 Method

2.2.1 Grouping

Set up three experimental groups:
( A)O Company (Lot. M****-02);
( B) M Company (Lot. *** 545);
( C) Bioteke (Cat. No.: AU46212).

2.2.2 DNA extraction

Soil DNA was extracted strictly according to the kit procedure , and the DNA extraction efficiency of different kits was compared. The extraction amount of DNA extracted by different kits and the feasibility of amplifying 16S rRNA gene were determined by gel electrophoresis and PCR amplification, respectively.

2.2.3 PCR amplification

The 16S rRNA gene in the soil and activated sludge microbial communities extracted after different treatments was PCR-amplified with the bacterial universal primer pair 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3'. The PCR cycle was: 94 ° C, 3 min, 1 cycle; 94 ° C, 30 s, 30 cycles; 55 ° C, 30 s, 30 cycles; 72 ° C, 1 min, 30 cycles; 72 ° C, 5 min, 1 cycle.

3. Results and analysis

3.1 Comparison of DNA concentration and integrity extracted

Table 1 results of soil genomic DNA ultra-micro spectrophotometer

Figure 1 Soil genomic DNA electropherogram

Fig.2 Electrophoresis map of 16S rRNA gene PCR amplification product of soil sample

3.2 Analysis of the comparison results of extracted DNA concentration and integrity

1. The DNA salinity extracted by group A is acceptable, but there are many PCR interference factors, and there is no band in PCR amplification;
2. There are only two kinds of extracted nucleic acids in the three soils of group B that meet the PCR conditions;
3. The soil DNA extracted from the soil extraction kit of group C has less salt residue and high purity. The extracted DNA has low PCR inhibitory factor, and the extracted DNA can be amplified normally, and the strip is single and clear.

4. References

[1] Torsvik V L. Microbial diversity and function in soil: from genes to ecosystems [J]. Current Opinion in Microbiology, 2002, 5(3): 240-245.

[2] Head IM, Saunders JR, Pickup R W. Microbial Evolution, Diversity, and Ecology: A Decade of Ribosomal RNA Analysis of Uncultivated Microorganisms [J]. Microbial Ecology, 1998, 35(1):1-21.

[3] Volossiouk T, Robb EJ, Nazar R N. Direct DNA extraction for PCR-mediated assays of soil organisms. [J]. Appl Environ Microbiol, 1995, 61(11): 3972-3976.

4] Tsai YL, Olson B H. Rapid method for direct extraction of DNA from soil and sediments. [J]. Applied & Environmental Microbiology, 1991, 57(4): 1070-4.

[5] Bürgmann H, Pesaro M, Widmer F, et al. A strategy for optimizing quality and quantity of DNA extracted from soil [J]. J Microbiol Methods, 2001, 45(1): 7-20.

[6] Wang Xiaobo, Tang Yuqiu, Wang Jinhua, et al. Isolation, purification and library construction of DNA in environmental samples[J]. Acta Microbiologica Sinica, 2001, 41(2): 133-140.

[7] Bourrain M, Achouak W, Urbain V, et al. DNA Extraction from Activated Sludges [J]. Current Microbiology, 1999, 38(6): 315-319.

[8] Lamontagne MG, Jr MF, Holden PA, et al. Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from compost for microbial community analysis. [J]. Journal of Microbiological Methods, 2002, 49(3): 255-264.