Method for extracting trace DNA in soil

Abstract

The invention relates to the technical field of biology, and particularly discloses a method for extracting trace DNA in soil, which comprises the following steps: carrying out mild incubation cracking on a soil sample by utilizing an extraction buffer solution and protease K, and releasing DNA adsorbed on the soil and DNA in cells in the soil sample into an extraction solution; according to the present invention, the phenol-chloroform-isoamyl alcohol nucleic acid extraction liquid is adopted to perform protein impurity removal, the ultrafiltration centrifuge tube is adopted to perform centrifugation, and filtration elution is performed to obtain the extraction liquid containing the target DNA, such that the trace environmental DNA or macro DNA in the soil can be efficiently extracted by using the method, and the method is especially suitable for the extraction of DNA in the extreme environment where the living beings are difficult to survive, and a technical support is provided for DNA-based biological research in an extreme habitat.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a method for extracting trace DNA from soil, and more specifically to a method for efficiently extracting trace DNA from multi-year deep frozen soil. Background Art

[0002] Soil is the most widely distributed habitat type in terrestrial ecosystems. Soil microbial communities are highly diverse and have complex ecological functions, including bacteria, archaea, fungi, viruses, protists, microscopic algae and rare microorganisms. These microbial communities have a profound impact on the soil biogeochemical cycle. Analyzing the functions of soil microbial communities and their dynamic changes will contribute to the material circulation and energy flow of the soil, and is of great significance to the understanding of ecological processes.

[0003] With the development of technologies such as high-throughput sequencing, metagenomic sequencing, and amplicon sequencing, soil microbial research no longer relies on laboratory isolation and pure culture technology, but can be analyzed through culture-free technology. Among them, complete and high-quality DNA extraction is the key prerequisite for the success of high-throughput sequencing and metagenomic research.

[0004] However, in extreme environments, there are fewer tolerable organisms and the DNA content is low; or in environmental DNA preserved in frozen soil environments, such as permafrost, its physical properties may make cell lysis and DNA release difficult, affecting the efficiency and quality of DNA extraction. At present, commercial kits are the mainstream method for extracting DNA, and their DNA extraction quality is acceptable, but such kits are generally suitable for samples with high DNA content, and the efficiency of DNA extraction is low. They cannot effectively extract DNA from samples with trace amounts of DNA. In addition, although the method of liquid nitrogen grinding combined with manual extraction of the extraction solution can obtain intracellular DNA more efficiently, it has a greater degree of damage to DNA with a higher degree of fragmentation, resulting in failure to extract trace amounts of DNA.

[0005] In summary, the current common methods for extracting microbial DNA from soil samples are prone to failure in extracting some trace DNA with low frequency and low abundance, and cannot effectively reflect the actual microbial composition in the soil, which has certain limitations. Summary of the invention

[0006] The purpose of this application is to overcome the shortcomings of the above-mentioned prior art and provide a method for extracting trace DNA in soil. The extraction method provided in this application can overcome the low DNA concentration in extreme habitats, or the shortening of DNA chains, severe DNA fragmentation, and physical damage to DNA caused by various reasons, and can extract high-abundance, high-quality, high-purity, and good-integrity DNA from soil samples as much as possible, effectively reducing DNA loss, thereby carrying out related high-throughput sequencing and metagenomic research.

[0007] To achieve the above purpose, the technical solution adopted by this application is: The present application provides a method for extracting trace DNA in soil, comprising the following steps: S1. Mix soil sample, extraction buffer and proteinase K, lyse and incubate overnight to obtain a preliminary lysate; S2, centrifuging the preliminary lysate obtained in step S1 for the first time, taking the supernatant and adding phenol-chloroform-isoamyl alcohol nucleic acid extraction solution to remove protein impurities, centrifuging for the second time, taking the supernatant, filtering and eluting through an ultrafiltration centrifuge tube, and obtaining a DNA extract; S3. Perform DNA quality detection and quantification on the DNA extract obtained in step S2, and then perform subsequent analysis or preserve the obtained soil DNA.

[0008] In the technical scheme of the present application, the present application uses extraction buffer and proteinase K to gently incubate and lyse the soil sample, and releases the DNA adsorbed on the soil and the DNA in the cells in the soil sample into the extract; protein impurities are removed by phenol-chloroform-isoamyl alcohol nucleic acid extraction solution, and then ultrafiltration centrifuge tubes are used for centrifugation, filtration and elution to obtain an extract containing the target DNA. The method of the present application can efficiently extract trace amounts of environmental DNA or macro-DNA in the soil, which is particularly suitable for the extraction of DNA in extreme environments where it is difficult for organisms to survive, and provides technical support for DNA-based biological research in extreme habitats.

[0009] If the total amount of DNA is insufficient, steps S1-S3 can be repeated until the requirements for subsequent analysis are met.

[0010] As a preferred embodiment of the method for extracting trace DNA in soil described in the present application, in step S1, the extraction buffer comprises sodium phosphate buffer, cell lysis solution and polyvinyl pyrrolidone; The sodium phosphate buffer comprises components of the following concentrations: 0.1M disodium hydrogen phosphate dodecahydrate and 0.1M sodium dihydrogen phosphate dihydrate, and the pH of the sodium phosphate buffer is 7.8-8.2; the cell lysate comprises components of the following concentrations: 0.5M tris(hydroxymethyl)aminomethane, 150mM sodium chloride, and 4% (w / v) sodium dodecyl sulfate.

[0011] This application uses a formulated sodium phosphate buffer, cell lysis solution and polyvinyl pyrrolidone combined with proteinase K, which can better incubate and lyse soil samples, release DNA adsorbed on the soil and DNA in cells in the soil sample into the extraction solution, and can cover DNA extraction to the greatest extent, reduce the possibility of DNA leakage, and effectively reflect the actual composition of microorganisms in the soil. The use of other extraction buffers will lead to protein contamination, ineffective removal of salt, and contamination by phenolic substances, resulting in poor DNA extraction or failure to successfully extract DNA.

[0012] As a preferred embodiment of the method for extracting trace DNA in soil described in the present application, the mixing ratio of the soil sample and the sodium phosphate buffer is 1g:1ml, and the mass ratio of the polyvinyl pyrrolidone and the soil sample is (0.0257-0.0515)g:1g; The mass ratio of the cell lysate to the soil sample is (25-50) μl:1 g; The mass ratio of the proteinase K to the soil sample is (46-48) μg:1g.

[0013] When the soil sample, sodium phosphate buffer, cell lysis solution, polyvinyl pyrrolidone and proteinase K are in the above ratio, the soil sample can be better lysed, and high-abundance, high-quality, high-purity and good-integrity DNA can be extracted from the soil sample as much as possible, effectively reducing DNA loss.

[0014] As a preferred embodiment of the method for extracting trace DNA in soil described in the present application, in step S2, the phenol-chloroform-isoamyl alcohol nucleic acid extraction solution is composed of a mixture of phenol, chloroform and isoamyl alcohol, and the volume ratio of the phenol, chloroform and isoamyl alcohol is 25:24:1.

[0015] The use of the phenol-chloroform-isoamyl alcohol nucleic acid extraction solution can better remove proteins in the preliminary lysate, reduce impurities in the DNA extraction solution, obtain DNA with better quality and higher integrity, and reduce the loss of DNA during the operation, thereby improving the DNA extraction efficiency.

[0016] As a preferred embodiment of the method for extracting trace DNA in soil described in the present application, in step S2, the conditions for the first centrifugation are: the centrifugal speed is 4000g, and the centrifugal speed is 9 to 15min; The conditions for the second centrifugation are: centrifugal speed is 4000g, and the centrifugal speed is 15 to 20min.

[0017] As a preferred embodiment of the method for extracting trace DNA in soil described in the present application, in step S2, the filtration and elution comprises the following steps: The supernatant was taken and filtered using a 10 kDa ultrafiltration centrifuge tube to obtain a target retentate containing DNA, and then the target retentate containing DNA was eluted using an eluent and centrifugally filtered again to obtain a DNA extract.

[0018] As a preferred embodiment of the method for extracting trace amounts of DNA in soil described in the present application, the elution solution comprises a 10 mM Tris-HCl buffer solution with a pH of 8.5.

[0019] As a preferred embodiment of the method for extracting trace DNA from soil described in the present application, in step S3, the quality of the obtained DNA is tested using an Agilent fragment analyzer system or a Nanodrop instrument.

[0020] As a preferred embodiment of the method for extracting trace DNA in soil described in the present application, in step S3, the obtained soil DNA is stored at -20°C.

[0021] As a preferred embodiment of the method for extracting trace amounts of DNA in soil described in the present application, the soil sample is a soil sample containing trace amounts of DNA that is difficult to extract, and the soil sample includes deep soil.

[0022] Compared with the prior art, this application has the following beneficial effects: The present application provides a method for extracting trace DNA in soil. The method of the present application can cover the extraction of DNA to the greatest extent, effectively reduce the pollution of protein, RNA and phenolic substances, enrich DNA to a certain extent, reduce the possibility of DNA leakage, and effectively reflect the actual composition of microorganisms in the soil; and the method for efficiently extracting DNA from permafrost provided by the present application is highly applicable, can extract trace and difficult-to-extract DNA from different types of habitats, extract DNA with good quality and high integrity, and lose relatively little DNA during operation, thereby effectively improving the extraction efficiency of DNA. DETAILED DESCRIPTION

[0023] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with specific embodiments.

[0024] In the following examples and comparative examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified, and the components and raw materials used in each parallel experiment are of the same type.

[0025] In the following examples, the sodium phosphate buffer contained the following components at the following concentrations: 0.1 M disodium hydrogen phosphate dodecahydrate and 0.1 M sodium dihydrogen phosphate dihydrate, and the pH of the sodium phosphate buffer was 8.0.

[0026] The cell lysis buffer contained the following components at the following concentrations: 0.5 M Tris (Tris), 150 mM sodium chloride, and 4% (w / v) sodium dodecyl sulfate (SDS).

[0027] The phenol-chloroform-isoamyl alcohol nucleic acid extract is composed of a mixture of phenol, chloroform and isoamyl alcohol, wherein the volume ratio of the phenol, chloroform and isoamyl alcohol is 25:24:1.

[0028] Example 1 This embodiment provides a method for extracting trace amounts of DNA in soil, comprising the following steps: S1. Add 5 ml sodium phosphate buffer, 0.129 g polyvinyl pyrrolidone, 125 μl cell lysis solution and 230 μg proteinase K to 5 g soil sample (deep permafrost sample from Bayi Glacier, Qinghai Province) and use 4.5 m·s in FastPrep instrument. -1 The cells were lysed at a speed of 40 s and then incubated at 37 °C with gentle rotation overnight to obtain a preliminary lysate. S2. Centrifuge the preliminary lysate obtained in step S1 for the first time at 4000g for 15 minutes, take the supernatant and transfer it to a new 15ml sterile centrifuge tube, add 10ml of phenol-chloroform-isoamyl alcohol nucleic acid extract to remove protein impurities, and then gently rotate and incubate at room temperature for 10 minutes. After the incubation is completed, take the supernatant for a second centrifugation at 4000g for 5 minutes, transfer the supernatant to a 10kDa centrifugal filter, and then centrifuge the sample at 4000g to concentrate the sample to 200μl to obtain the target retentate containing DNA, and elute the target retentate containing DNA with 1.0ml of elution solution, eluting twice. Centrifuge the eluted sample again to concentrate the sample to 200μl to obtain the final DNA extract.

[0029] S3. Perform DNA quality detection (using Agilent fragment analyzer system or Nanodrop and other instruments to detect the quality of the obtained DNA) and quantification on the final DNA extract, and store the DNA extract at -20°C.

[0030] The results of deep frozen soil extraction using the extraction method of Example 1 are shown in Table 1.

[0031] Table 1 Results of deep frozen soil extraction using the extraction method of Example 1 Example 2 DNA was extracted from the permafrost sample (BY-1-1.8m) from the Bayi Glacier in Qinghai according to the extraction method of Example 1, wherein the amounts of cell lysate used were 125 μl, 187.5 μl and 250 μl, respectively. The obtained DNA was quality tested and quantified using a Qubit instrument. The DNA extraction results are shown in Table 2.

[0032] Table 2 Results of extracting deep frozen soil by adjusting the amount of cell lysate using the extraction method of Example 1 Example 3 DNA was extracted from a frozen soil sample (BY-1-1.8m) from the Bayi Glacier in Qinghai according to the extraction method of Example 1, wherein the amount of cell lysate used was 250 μl, the amount of polyvinyl pyrrolidone added was 0.257 g, and the obtained DNA was quality tested and quantified using a Qubit instrument. The DNA extraction results are shown in Table 3.

[0033] Table 3 Results of extracting deep frozen soil by adjusting the concentration of polyvinyl pyrrolidone using the extraction method of Example 1 Comparative Example 1 Comparative Example 1: The Tiangen Magnetic Bead Soil Genomic DNA Extraction Kit (DP712) was used to extract DNA from deep frozen soil samples from the Bayi Glacier in Qinghai according to the instructions provided.

[0034] Take 5-10 samples of each deep frozen soil sample, each weighing 0.25 g, and extract them according to the instructions provided. After the extraction, concentrate and combine the obtained DNA samples. The extraction results are shown in Table 4 below: Table 4 Results of deep frozen soil extraction using Tiangen DNA extraction kit Comparative Example 2 Comparative Example 2: The CTAB extraction method was used to extract DNA from deep frozen soil samples from the Bayi Glacier in Qinghai.

[0035] The specific implementation methods are as follows: 1) Sample: Pipette 1000 μl of CTAB lysis buffer containing lysozyme into a 2.0 ml EP tube, add an appropriate amount of sample to the lysis buffer, incubate in a 65°C water bath, and invert several times to mix well to ensure that the sample is fully lysed.

[0036] 2) Centrifuge and take the supernatant, add phenol (pH 8.0): chloroform: isoamyl alcohol (25:24:1), invert to mix, and centrifuge at 12000 rpm for 10 min.

[0037] 3) Take the supernatant, add chloroform:isoamyl alcohol (24:1), mix by inversion, and centrifuge at 12000 rpm for 10 min.

[0038] 4) Pipette the supernatant into a 1.5 ml centrifuge tube, add isopropanol, shake up and down, and precipitate at -20℃.

[0039] 5) Centrifuge at 12000rpm for 10 minutes, pour out the liquid, be careful not to pour out the precipitate. Wash twice with 1ml 75% ethanol. The remaining small amount of liquid can be collected by centrifugation again and then sucked out with a pipette.

[0040] 6) Blow dry on a clean bench or air dry at room temperature (the DNA sample should not be too dry, otherwise it will be difficult to dissolve), add sterile water to dissolve the DNA sample, and incubate at 55-60℃ for 10 minutes to aid dissolution if necessary.

[0041] 7) Add 1 μl of RNase A to digest RNA and incubate at 37°C for 15 min.

[0042] Take 5-10 samples of each deep frozen soil sample, each weighing 0.25 g, and extract them according to the instructions provided. After the extraction, concentrate and combine the obtained DNA samples. The extraction results are shown in Table 5 below: Table 5 Results of deep frozen soil extraction using CTAB extraction method Comparative Example 3 Comparative Example 3 is a method of extracting DNA from deep frozen soil samples from the Bayi Glacier in Qinghai using a liquid nitrogen grinding method combined with a QIAGEN kit (all reagents used below are from the QIAGEN kit).

[0043] The specific implementation plan is as follows: 1. Liquid nitrogen grinding: 1) Add 16.5 ml of the extraction buffer in Example 1 to a sterile 50 ml centrifuge tube (tube A) in advance. Add sterile quartz sand; take out about 5 g of the sample, then add an appropriate amount of liquid nitrogen in a mortar; repeat freezing and grinding.

[0044] 2) Transfer the ground sample to a 50 ml centrifuge tube A, add 61 μl of proteinase K (10 mg / ml), and mix gently.

[0045] 2. Crude DNA extraction: 3) Place tube A in a 37°C water bath and incubate for 30 min, add 1.83 ml 20% SDS, and mix gently. Place tube A in a 65°C water bath and incubate for 2 h; centrifuge at 6000 g, 25°C, for 20 min; transfer the supernatant to tube B.

[0046] 4) Add an equal volume of isoamyl alcohol-chloroform (1:24) to tube B and mix for 5-10 minutes; centrifuge tube B at 3700g, 25°C for 20 minutes.

[0047] 5) Transfer the upper aqueous phase to a 50 mL loak ridge tube C and add 0.6 volumes of isopropanol; place tube C at -20°C overnight.

[0048] 2. Kit for DNA purification: 6) Place tube C in a 37°C water bath. Centrifuge at 15,000 g, 25°C for 20 min; transfer the supernatant to tube D.

[0049] 7) Add 1 ml of 70% ethanol to tube D to wash the precipitated DNA particles, and transfer the DNA and ethanol to a centrifuge tube.

[0050] 8) Add 680 μl CD1 solution to dissolve DNA and vortex to mix; centrifuge at 10,000 g for 1 min; transfer 650 μl supernatant to a 2 ml centrifuge tube; add 200 μl CD2 solution.

[0051] 9) Centrifuge at 15000g for 2 min; transfer 700μl supernatant to a 2 ml centrifuge tube; add 600μl CD3 solution; add 650μl lysate to the MB Spin Column; centrifuge at 15000g for 2 min.

[0052] 10) Transfer the MB Spin Column to a new 2 ml centrifuge tube; add 500 μl of solution EA to the MB Spin Column, let it stand for 5 min, centrifuge at 15,000 g for 2 min, and discard the liquid in the tube. Put the MB Spin Column back into the centrifuge tube, add 500 μl of solution C5, let it stand for 5 min, centrifuge at 15,000 g for 2 min, discard the liquid in the tube, and place the MB Spin Column in a 2 ml centrifuge tube.

[0053] 11) Centrifuge at 16000g for 2 min, transfer the MB Spin Column to a 1.5 ml centrifuge tube; add 100 μl of solution C6 to the center of the MB Spin Column, let stand for 5 min, and centrifuge at 15000g for 2 min. Discard the MB Spin Column.

[0054] The DNA samples obtained after extraction were tested and the results are shown in Table 6 below: Table 6 Extraction results of deep frozen soil extracted by liquid nitrogen grinding + QIAGEN kit Comparative Example 4 Similar to Example 1, the difference of Comparative Example 4 is that FastDNA TM The SPIN Filter of the SPIN Kit for Soil kit replaced the 10 kDa ultrafiltration centrifuge tube in Example 1. The remaining steps were the same as in Example 1. DNA was extracted from the frozen soil sample (BY-1-1.8m) from the Bayi Glacier in Qinghai. The test results are shown in Table 7 below: Table 7 Using FastDNA TM The extraction results of deep frozen soil were extracted by replacing the ultrafiltration step in Example 1 with the SPIN Kit In combination with Example 1, Comparative Example 1, Comparative Example 2 and Comparative Example 3, as well as Example 2, Example 3 and Comparative Example 4, the DNA samples extracted by the extraction method of the present application are superior to commercial kits and traditional CTAB, liquid nitrogen grinding extraction methods in terms of DNA concentration and total DNA amount, and after testing, meet the requirements of high-throughput sequencing. This provides technical support for the extraction of difficult-to-extract trace DNA in extreme habitats and related research.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. A method for extracting trace DNA from soil, characterized in that: The following steps are involved: S1. Mix soil sample, extraction buffer and proteinase K, lyse and incubate overnight to obtain a preliminary lysate; S2, centrifuging the preliminary lysate obtained in step S1 for the first time, taking the supernatant and adding phenol-chloroform-isoamyl alcohol nucleic acid extraction solution to remove protein impurities, centrifuging for the second time, taking the supernatant, filtering and eluting through an ultrafiltration centrifuge tube, and obtaining a DNA extract; S3. Perform DNA quality detection and quantification on the DNA extract obtained in step S2, and then perform subsequent analysis or preserve the obtained soil DNA.

2. The method for extracting trace DNA in soil according to claim 1, characterized in that: In step S1, the extraction buffer comprises sodium phosphate buffer, cell lysis solution and polyvinyl pyrrolidone; The sodium phosphate buffer contains the following components at the following concentrations: 0.1M disodium hydrogen phosphate dodecahydrate and 0.1M sodium dihydrogen phosphate dihydrate, the pH of the sodium phosphate buffer is 7.8-8.2; the cell lysate contains the following components in the following concentrations: 0.5 M Tris, 150 mM sodium chloride, 4% (w / v) sodium dodecyl sulfate.

3. The method for extracting trace DNA from soil as claimed in claim 2, characterized in that: The mixing ratio of the soil sample and the sodium phosphate buffer is 1 g:1 ml, the mass ratio of the polyvinyl pyrrolidone to the soil sample is (0.0257-0.0515) g:1 g; the mass ratio of the cell lysate to the soil sample is (25-50) μl:1 g; The mass ratio of the proteinase K to the soil sample is (46-48) μg:1g.

4. The method for extracting trace DNA in soil according to claim 1, characterized in that: In step S2, the phenol-chloroform-isoamyl alcohol nucleic acid extract is composed of a mixture of phenol, chloroform and isoamyl alcohol, and the volume ratio of the phenol, chloroform and isoamyl alcohol is 25:24:

1.

5. The method for extracting trace DNA in soil according to claim 1, characterized in that: In step S2, the conditions for the first centrifugation are: a centrifugal speed of 4000 g and a centrifugal speed of 9 to 15 min; The conditions for the second centrifugation are: centrifugal speed is 4000g, and the centrifugal speed is 15 to 20min.

6. The method for extracting trace DNA in soil according to claim 1, characterized in that: In step S2, filtering and eluting comprises the following steps: The supernatant was taken and filtered using a 10 kDa ultrafiltration centrifuge tube to obtain a target retentate containing DNA, and then the target retentate containing DNA was eluted using an eluent and centrifugally filtered again to obtain a DNA extract.

7. The method for extracting trace DNA in soil according to claim 6, characterized in that: The eluent included 10 mM Tris-HCl buffer at pH=8.

5.

8. The method for extracting trace DNA in soil according to claim 1, characterized in that: In step S3, the quality of the obtained DNA is tested using an Agilent fragment analyzer system or a Nanodrop instrument.

9. The method for extracting trace DNA in soil according to claim 1, characterized in that: In step S3, the obtained soil DNA is stored at -20°C.

10. The method for extracting trace DNA in soil according to claim 1, characterized in that: The soil sample is a soil sample containing trace or small amounts of DNA that is difficult to extract.

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