A method for extracting trace DNA from soil

Abstract

This application relates to the field of biotechnology, specifically disclosing a method for extracting trace DNA from soil. This application utilizes extraction buffer and proteinase K to gently incubate and lyse soil samples, releasing DNA adsorbed on the soil and intracellular DNA into the extraction solution. Protein removal is performed using phenol-chloroform-isoamyl alcohol nucleic acid extraction buffer, followed by centrifugation, filtration, and elution using ultrafiltration centrifuge tubes to obtain an extract containing the target DNA. This method can efficiently extract trace amounts of environmental DNA or metaDNA from soil, and is particularly suitable for DNA extraction in extreme environments where biological survival is difficult, providing technical support for DNA-based biological research in extreme habitats.

Description

Technical Field

[0001] This 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 deep permafrost. Background Technology

[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, protozoa, microalgae, and rare microorganisms. These microbial communities have a profound impact on soil biogeochemical cycles. Analyzing the functions and dynamic changes of soil microbial communities will help with soil material cycling and energy flow, and is of great significance for understanding ecological processes.

[0003] With the development of technologies such as high-throughput sequencing, metagenomic sequencing, and amplicon sequencing, research on soil microorganisms no longer relies on laboratory isolation and pure culture techniques, but can be conducted through culture-free techniques. Among these, complete, high-quality DNA extraction is a crucial prerequisite for the success of high-throughput sequencing and metagenomic research.

[0004] However, in extreme environments, the number of tolerant organisms is limited, resulting in lower DNA content. Alternatively, environmental DNA preserved in permafrost environments, such as deep permafrost, may present challenges due to its physical properties, making cell lysis and DNA release difficult, thus affecting the efficiency and quality of DNA extraction. Currently, commercially available kits are the mainstream method for DNA extraction, offering acceptable quality. However, these kits are generally suitable for samples with high DNA content, but their extraction efficiency is low, and they are ineffective for extracting trace amounts of DNA. Furthermore, while liquid nitrogen grinding combined with manual extraction with extraction buffer can efficiently obtain intracellular DNA, it significantly damages highly fragmented DNA, leading to extraction failures for trace amounts of DNA.

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

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

[0007] To achieve the above objectives, the technical solution adopted in this application is as follows:

[0008] This application provides a method for extracting trace amounts of DNA from soil, comprising the following steps:

[0009] S1. Mix the soil sample, extraction buffer, and proteinase K, and incubate overnight to obtain a preliminary lysis buffer;

[0010] S2. Centrifuge the preliminary lysis buffer obtained in step S1 for the first time, take the supernatant and add phenol-chloroform-isoamyl alcohol nucleic acid extraction buffer to remove protein impurities, centrifuge for the second time, take the supernatant and filter and elute through an ultrafiltration centrifuge tube to obtain DNA extraction solution.

[0011] S3. Perform DNA quality testing and quantification on the DNA extract obtained in step S2, and then conduct further analysis or preserve the obtained soil DNA.

[0012] In the technical solution of this application, soil samples are gently incubated and lysed using extraction buffer and proteinase K to release DNA adsorbed on the soil and DNA from cells into the extraction solution. Proteins are removed using phenol-chloroform-isoamyl alcohol nucleic acid extraction solution, and then the sample is centrifuged, filtered, and eluted using an ultrafiltration centrifuge tube to obtain an extraction solution containing the target DNA. The method of this application can efficiently extract trace amounts of environmental DNA or metaDNA from soil, and is especially suitable for DNA extraction in extreme environments where biological survival is difficult, providing technical support for DNA-based biological research in extreme habitats.

[0013] If the total amount of DNA is insufficient, steps S1-S3 can be repeated until the requirements for subsequent analysis are met.

[0014] In a preferred embodiment of the method for extracting trace DNA from soil as described in this application, in step S1, the extraction buffer includes sodium phosphate buffer, cell lysis buffer, and polyvinylpyrrolidone.

[0015] 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, and the pH of the sodium phosphate buffer is 7.8–8.2; the cell lysis buffer contains the following components at the following concentrations: 0.5M tris(hydroxymethyl)aminomethane, 150mM sodium chloride, and 4% (w / v) sodium dodecyl sulfate.

[0016] This application utilizes a formulated sodium phosphate buffer, cell lysis buffer, and polyvinylpyrrolidone combined with proteinase K to better incubate and lyse soil samples. This releases DNA adsorbed on the soil surface and intracellular DNA into the extraction solution, maximizing DNA extraction coverage, reducing the possibility of missed DNA detection, and effectively reflecting the actual microbial composition in the soil. Using other extraction buffers can lead to protein contamination, ineffective salt removal, and phenolic contamination, resulting in poor DNA extraction or even failure to extract DNA.

[0017] In a preferred embodiment of the method for extracting trace DNA from soil as described in this application, the soil sample and sodium phosphate buffer are mixed in a ratio of 1g:1ml, and the mass ratio of polyvinylpyrrolidone to soil sample is (0.0257~0.0515)g:1g.

[0018] The mass ratio of the cell lysate to the soil sample was (25–50) μl: 1 g;

[0019] The mass ratio of proteinase K to soil sample was (46–48) μg: 1g.

[0020] When soil samples, sodium phosphate buffer, cell lysis buffer, polyvinylpyrrolidone, and proteinase K are used in the above ratio, soil samples can be lysed more effectively, and high-abundance, high-quality, high-purity, and well-intact DNA can be extracted from the soil samples, effectively reducing DNA loss.

[0021] In a preferred embodiment of the method for extracting trace DNA from soil as described in this application, in step S2, the phenol-chloroform-isoamyl alcohol nucleic acid extraction solution is composed of a mixture of phenol, chloroform and isoamyl alcohol, wherein the volume ratio of phenol, chloroform and isoamyl alcohol is 25:24:1.

[0022] Using the above-mentioned phenol-chloroform-isoamyl alcohol nucleic acid extraction solution can better remove proteins from the initial lysis buffer, reduce impurities in the DNA extraction solution, and obtain DNA of better quality and higher integrity. Less DNA is lost during the operation, thus improving the DNA extraction efficiency.

[0023] As a preferred embodiment of the method for extracting trace DNA from soil described in this application, in step S2, the conditions for the first centrifugation are: centrifugation speed of 4000g and centrifugation speed of 9-15min.

[0024] The conditions for the second centrifugation were: centrifugation speed of 4000g and centrifugation speed of 15-20min.

[0025] In a preferred embodiment of the method for extracting trace DNA from soil as described in this application, step S2, filtration and elution, includes the following steps:

[0026] Collect the supernatant and filter it using a 10kDa ultrafiltration centrifuge tube to obtain the target intercept containing DNA. Then, elute the target intercept containing DNA with elution buffer and centrifuge and filter again to obtain the DNA extract.

[0027] As a preferred embodiment of the method for extracting trace DNA from soil as described in this application, the eluent comprises a 10 mM Tris-HCl buffer at pH 8.5.

[0028] In a preferred embodiment of the method for extracting trace DNA from soil as described in this application, in step S3, the obtained DNA is subjected to quality testing using an Agilent fragment analyzer system or a Nanodrop instrument.

[0029] In a preferred embodiment of the method for extracting trace DNA from soil as described in this application, the obtained soil DNA in step S3 is stored at -20°C.

[0030] As a preferred embodiment of the method for extracting trace DNA from soil described in this application, the soil sample is a soil sample containing trace or minute amounts of difficult-to-extract DNA, and the soil sample includes deep soil.

[0031] Compared with the prior art, this application has the following advantages:

[0032] This application provides a method for extracting trace DNA from soil. The method described herein can maximize DNA extraction coverage, effectively reduce contamination by proteins, RNA, and phenolic substances, enrich DNA to a certain extent, reduce the possibility of missed DNA detection, and effectively reflect the actual microbial composition in the soil. Furthermore, the efficient method for extracting DNA from deep permafrost provided in this application has strong applicability, can extract trace and difficult-to-extract DNA from different types of habitats, extracts high-quality and high-integrity DNA, and has relatively little DNA loss during operation, effectively improving DNA extraction efficiency. Detailed Implementation

[0033] To better illustrate the purpose, technical solution, and advantages of this application, the following will provide further explanation of this application in conjunction with specific embodiments.

[0034] In the following examples and comparative examples, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified. Furthermore, the raw materials used in each parallel experiment are the same.

[0035] In the following examples, the sodium phosphate buffer contains 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 is 8.0.

[0036] The cell lysate contained the following components at the following concentrations: 0.5 M tris(hydroxymethyl)aminomethane (Tris), 150 mM sodium chloride, and 4% (w / v) sodium dodecyl sulfate (SDS).

[0037] The phenol-chloroform-isoamyl alcohol nucleic acid extraction solution is composed of a mixture of phenol, chloroform and isoamyl alcohol, wherein the volume ratio of phenol, chloroform and isoamyl alcohol is 25:24:1.

[0038] Example 1

[0039] This embodiment provides a method for extracting trace DNA from soil, including the following steps:

[0040] S1. Add 5 ml of sodium phosphate buffer, 0.129 g of polyvinylpyrrolidone, 125 μl of cell lysis buffer, and 230 μg of proteinase K to a 5 g soil sample (deep permafrost sample from the Bayi Glacier in Qinghai). Run the mixture on a FastPrep instrument at 4.5 m·s. -1 The solution was lysed at a rate of 40 s, and then gently rotated and incubated overnight at 37 °C to obtain a preliminary lysate.

[0041] S2. Centrifuge the preliminary lysis buffer obtained in step S1 at 4000g for 15 min. Transfer the supernatant to a new 15ml sterile centrifuge tube, add 10ml of phenol-chloroform-isoamyl alcohol nucleic acid extraction buffer for protein removal, and then gently incubate at room temperature for 10 min. After incubation, centrifuge the supernatant a second time at 4000g for 5 min. Transfer the supernatant to a 10kDa centrifugal filter, then concentrate the sample to 200μl by centrifugation at 4000g to obtain the target DNA-containing precipitate. Elute the target DNA-containing precipitate with 1.0ml of elution buffer twice. Centrifuge the eluted sample again to concentrate it to 200μl to obtain the final DNA extract.

[0042] S3. Perform DNA quality testing (using an Agilent fragment analyzer system or an instrument such as Nanodrop) and quantification on the obtained DNA extract, and store the DNA extract at -20°C.

[0043] The results of extracting deep permafrost using the extraction method of Example 1 are shown in Table 1.

[0044] Table 1 shows the results of extraction of deep permafrost using the extraction method of Example 1.

[0045]

[0046] Example 2

[0047] DNA was extracted from a permafrost sample (BY-1-1.8m) from the Bayi Glacier in Qinghai Province according to the extraction method in Example 1. The amounts of cell lysis buffer used were 125 μl, 187.5 μl, and 250 μl, respectively. The quality of the obtained DNA was detected and quantified using a Qubit instrument. The DNA extraction results are shown in Table 2.

[0048] Table 2 shows the results of extracting deep permafrost using the extraction method of Example 1 with adjustments to the amount of cell lysis buffer.

[0049] Example 3

[0050] DNA was extracted from a permafrost sample (BY-1-1.8m) from the Bayi Glacier in Qinghai Province according to the extraction method in Example 1. The amount of cell lysis buffer used was 250 μl, and the amount of polyvinylpyrrolidone added was 0.257 g. The quality of the obtained DNA was detected and quantified using a Qubit instrument. The DNA extraction results are shown in Table 3.

[0051] Table 3 shows the results of extracting deep permafrost using the extraction method of Example 1 with adjusted polyvinylpyrrolidone concentration.

[0052] Comparative Example 1

[0053] Comparative Example 1 used the Tiangen Magnetic Bead Soil Genomic DNA Extraction Kit (DP712) to extract DNA from deep permafrost samples from the Bayi Glacier in Qinghai Province, following the provided instructions.

[0054] Take 5-10 samples from each deep permafrost sample, 0.25g each, and extract DNA according to the provided instructions. After extraction, concentrate and combine the obtained DNA samples. The extraction results are shown in Table 4 below:

[0055] Table 4 shows the results of DNA extraction from deep permafrost using the Tiangen DNA extraction kit.

[0056]

[0057] Comparative Example 2

[0058] Comparative Example 2 used the CTAB extraction method to extract DNA from deep permafrost samples from the Bayi Glacier in Qinghai.

[0059] The specific implementation method is as follows:

[0060] 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 and mix several times during the process to ensure that the sample is fully lysed.

[0061] 2) Centrifuge and collect the supernatant. Add phenol (pH 8.0): chloroform: isoamyl alcohol (25:24:1), invert and mix well. Centrifuge at 12000 rpm for 10 min.

[0062] 3) Take the supernatant, add chloroform:isoamyl alcohol (24:1), mix by inverting, and centrifuge at 12000 rpm for 10 min.

[0063] 4) Transfer the supernatant to a 1.5ml centrifuge tube, add isopropanol, shake up and down, and precipitate at -20℃.

[0064] 5) Centrifuge at 12000 rpm for 10 minutes, pour out the liquid, being careful not to pour out the precipitate. Wash twice with 1 ml of 75% ethanol. The remaining small amount of liquid can be collected by centrifugation again and then aspirated with a pipette tip.

[0065] 6) Dry the DNA sample in a clean bench or at room temperature (do not dry the DNA sample too much, otherwise it will be difficult to dissolve). Add sterile water to dissolve the DNA sample. If necessary, incubate at 55-60℃ for 10 minutes to aid dissolution.

[0066] 7) Add 1 μl of RNase A to digest the RNA and incubate at 37°C for 15 min.

[0067] Take 5-10 samples from each deep permafrost sample, 0.25g each, and extract DNA according to the provided instructions. After extraction, concentrate and combine the obtained DNA samples. The extraction results are shown in Table 5 below:

[0068] Table 5 shows the results of extraction of deep permafrost using the CTAB extraction method.

[0069]

[0070] Comparative Example 3

[0071] Comparative Example 3 describes the extraction of DNA from deep permafrost samples from the Bayi Glacier in Qinghai Province using a combination of liquid nitrogen grinding and a QIAGEN kit (all reagents used below are from the QIAGEN kit).

[0072] The specific implementation plan is as follows:

[0073] 1. Liquid nitrogen grinding:

[0074] 1) Add 16.5 ml of the extraction buffer from Example 1 to a sterile 50 ml centrifuge tube (tube A) in advance. Add sterile quartz sand; take out about 5 g of sample, and then add an appropriate amount of liquid nitrogen to a mortar; repeat freezing and grinding.

[0075] 2) Transfer the ground sample to a 50ml centrifuge tube A, add 61μl proteinase K (10mg / ml), and mix gently.

[0076] 2. Crude DNA extraction:

[0077] 3) Incubate tube A in a 37°C water bath for 30 minutes, add 1.83 ml of 20% SDS, and mix gently. Incubate tube A in a 65°C water bath for 2 hours; centrifuge at 6000 g, 25°C for 20 minutes; transfer the supernatant to tube B.

[0078] 4) Add an equal volume of isoamyl alcohol-chloroform (1:24) to tube B and mix for 5-10 minutes; place tube B at 3700g, 25℃ and centrifuge for 20 minutes.

[0079] 5) Transfer the upper aqueous phase to a 50ml loak ridge tube C and add 0.6 times the volume of isopropanol; place tube C at -20°C overnight.

[0080] 2. DNA purification using the kit:

[0081] 6) Place tube C in a 37°C water bath. Centrifuge at 15000g, 25°C for 20 minutes; transfer the supernatant to tube D.

[0082] 7) Add 1 ml of 70% ethanol to tube D to wash away the precipitated DNA particles, and transfer the DNA and ethanol to a centrifuge tube.

[0083] 8) Add 680 μl of CD1 solution to dissolve the DNA, vortex to mix; centrifuge at 10000g for 1 min; transfer 650 μl of supernatant to a 2 ml centrifuge tube; add 200 μl of CD2 solution.

[0084] 9) Centrifuge at 15000g for 2 min; transfer 700 μl of supernatant to a 2 ml centrifuge tube; add 600 μl of solution CD3; add 650 μl of lysis buffer to the MB Spin Column; centrifuge at 15000g for 2 min.

[0085] 10) Transfer the MB Spin Column to a new 2ml centrifuge tube; add 500μl of solution EA to the MB Spin Column, let stand for 5min, centrifuge at 15000g for 2min, and discard the liquid in the tube. Put the MB Spin Column back into the centrifuge tube, add 500μl of solution C5, let stand for 5min, centrifuge at 15000g for 2min, discard the liquid in the tube, and put the MB Spin Column into the 2ml centrifuge tube.

[0086] 11) Centrifuge at 16000g for 2 min, then 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.

[0087] The DNA samples obtained after extraction were tested, and the results are shown in Table 6 below:

[0088] Table 6 shows the extraction results of deep permafrost using liquid nitrogen grinding and the QIAGEN kit.

[0089]

[0090] Comparative Example 4

[0091] Similar to Example 1, the difference in Comparative Example 4 is that FastDNA was used. TM The SPIN Kit for Soil uses a SPIN Filter instead of the 10kDa ultrafiltration centrifuge tube in Example 1. The remaining steps are the same as in Example 1. DNA extraction was performed on a permafrost sample (BY-1-1.8m) from the Bayi Glacier in Qinghai Province. The results are shown in Table 7 below.

[0092] Table 7 uses FastDNA. TM Extraction results of deep permafrost using the SPIN Kit to replace the ultrafiltration step in Example 1

[0093]

[0094] In conjunction with Examples 1, 2, and 3, as well as Examples 2, 3, and 4, the DNA samples extracted using the method of this application showed superior DNA concentration and total amount compared to commercially available kits and traditional methods such as CTAB and liquid nitrogen grinding extraction. Furthermore, the samples met the requirements for high-throughput sequencing. This provides technical support for the extraction of trace amounts of DNA that are difficult to extract in extreme habitats and for related research.

[0095] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.

Claims

1. A method for extracting trace amounts of DNA from soil, characterized in that, Includes the following steps: S1. Mix the soil sample, extraction buffer, and proteinase K, and incubate overnight to obtain a preliminary lysis buffer; S2. Centrifuge the preliminary lysis buffer obtained in step S1 for the first time, take the supernatant and add phenol-chloroform-isoamyl alcohol nucleic acid extraction buffer to remove protein impurities, centrifuge for the second time, take the supernatant and filter and elute through an ultrafiltration centrifuge tube to obtain DNA extraction solution. S3. Perform DNA quality testing and quantification on the DNA extract obtained in step S2, and then conduct further analysis or preserve the obtained soil DNA. In step S1, the extraction buffer consists of sodium phosphate buffer, cell lysis buffer and polyvinylpyrrolidone. The sodium phosphate buffer solution consists of the following components at the following concentrations: 0.1M disodium hydrogen phosphate dodecahydrate and 0.1M sodium dihydrogen phosphate dihydrate, with a sodium phosphate buffer solution at pH 7.8–8.2; The cell lysis buffer consists of the following components at the following concentrations: 0.5M tris(hydroxymethyl)aminomethane, 150mM sodium chloride, 4% (w / v) sodium dodecyl sulfate; The soil sample and sodium phosphate buffer were mixed at a ratio of 1g:1ml, and the mass ratio of polyvinylpyrrolidone to soil sample was (0.0257~0.0515)g:1g. The mass ratio of the cell lysate to the soil sample was (25~50) μl:1g; The mass ratio of proteinase K to soil sample was (46~48) μg:1g; In step S2, filtration and elution include the following steps: Take the supernatant and filter it using a 10kDa ultrafiltration centrifuge tube to obtain the target residue containing DNA. Then, elute the target residue containing DNA with elution buffer and centrifuge and filter again to obtain the DNA extract. The elution buffer is a 10 mM Tris-HCl buffer solution with pH = 8.

5.

2. The method for extracting trace DNA from soil as described in claim 1, characterized in that, 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 phenol, chloroform and isoamyl alcohol is 25:24:

1.

3. The method for extracting trace DNA from soil as described in claim 1, characterized in that, In step S2, the conditions for the first centrifugation are: centrifugation speed of 4000g and centrifugation speed of 9~15 min; The conditions for the second centrifugation were: centrifugation speed of 4000g and centrifugation speed of 15~20 min.

4. The method for extracting trace DNA from soil as described in 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.

5. The method for extracting trace DNA from soil as described in claim 1, characterized in that, In step S3, the obtained soil DNA is stored at -20°C.

6. The method for extracting trace DNA from soil as described in claim 1, characterized in that, The soil sample was a soil sample containing trace or minute amounts of DNA that was difficult to extract.

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