Extraction method of metagenome DNA of ancient dental calculus

Abstract

The invention provides a metagenome DNA extraction method of ancient dental calculus, and belongs to the technical field of genetic engineering. The method comprises the steps of sample pretreatment, DNA extraction, purification, quality inspection and the like, and by optimizing an extraction reagent and method, the completeness of DNA can be maintained, meanwhile, exogenous pollution can be reduced to the maximum extent, and the DNA extraction efficiency is improved. The method is suitable for different types of ancient dental calculus samples, high-quality metagenome DNA can be obtained in a short time, and a solid foundation is provided for subsequent genome sequencing and analysis. According to the method, the high-quality metagenome DNA can be extracted from the ancient dental calculus within a short time, the extraction efficiency is remarkably improved, the pollution rate is remarkably reduced, and the method is suitable for various types of ancient dental calculus samples. The invention not only provides a powerful tool for scientific research, but also lays a foundation for genome sequencing and functional analysis of ancient dental calculus.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and specifically relates to a metagenomic DNA extraction method for ancient tartars. Background Art

[0002] The study of ancient microbial remains is of great significance for understanding the evolution of early life on Earth, environmental changes, and ancient ecosystems. The analysis of metagenomic DNA in these remains can reveal the composition and functional characteristics of microbial communities, thereby helping scientists reconstruct the biodiversity and ecological networks of ancient environments. However, since the DNA in ancient samples is usually severely degraded, has extremely low content, and is easily contaminated, how to efficiently extract metagenomic DNA has become a major challenge in scientific research.

[0003] Some existing extraction methods often have the following problems: 1. Insufficient extraction efficiency: Many methods are difficult to extract sufficient amounts of DNA from low concentrations of ancient samples, resulting in difficulty in subsequent analysis.

[0004] 2. Severe DNA contamination: Since ancient samples are susceptible to exogenous DNA contamination, existing methods often find it difficult to distinguish between target DNA in the sample from exogenous DNA.

[0005] 3. Poor DNA quality: The extracted DNA is often severely fragmented and cannot meet the requirements of high-throughput sequencing.

[0006] In the study of extracting ancient DNA from ancient dental calculus, it was found that dental calculus, as a research material, contains a large amount of DNA from oral microorganisms and food, and is an important resource for studying the oral flora and dietary structure of ancient humans. However, due to the complex structure of dental calculus samples, the DNA extraction process requires longer digestion and more complex purification operations to remove impurities that affect subsequent reactions. Although some existing methods, such as the phenol-chloroform method combined with commercial purification columns, can extract DNA from ancient dental calculus, they still have the above-mentioned problems of low extraction efficiency and severe DNA contamination.

[0007] Therefore, there is a need for an efficient extraction method that can improve extraction efficiency and reduce exogenous contamination while ensuring DNA integrity. Summary of the invention

[0008] The purpose of the present invention is to provide an efficient and low-pollution method for extracting metagenomic DNA from ancient dental calculus, aiming to overcome the shortcomings of the prior art and provide a reliable solution by optimizing each extraction step.

[0009] To achieve the above objectives, the specific technical solutions adopted by the present invention are: A method for extracting metagenomic DNA from ancient dental calculus comprises the following steps: (1) Sample pretreatment: Use ethanol to clean the tooth surface and scrape off the dental calculus on the tooth surface; (2) Sample lysis: Add lysis solution to the dental calculus sample and then perform low-frequency crushing; (3) Sample digestion; (4) DNA extraction.

[0010] Furthermore, in step (1), the ethanol concentration used is 75%, and all instruments and equipment used are sterilized by ultraviolet light.

[0011] Furthermore, in step (2), the lysis solution is prepared by adding 10 μl of proteinase K (20 mg / ml) to 990 μl of 0.5 M EDTA solution; the cells are disrupted at a low frequency of 60 rpm until no debris is visible to the naked eye; because ancient DNA fragments are short and easily damaged, conventional cell disruption methods are not used.

[0012] Furthermore, in step (3), the sample is placed in a metal bath with a shaking function, and digested overnight at 55°C and 1000 rpm to ensure that the sample is suspended and mixed; after digesting overnight, the sample is placed in a rotary mixer and digested for 48 hours at room temperature.

[0013] Furthermore, in step (4), the digested sample was centrifuged at 12,000 rpm for 1 minute, 950 μl of the supernatant was aspirated into a new 2 ml centrifuge tube, 1 ml of a mixture of phenol: chloroform: isopropanol (25:24:1) was added, vortexed and centrifuged at 12,000 rpm for 1 minute, the supernatant was transferred to a new 2 ml centrifuge tube, and this process was repeated twice. 1 ml of chloroform was added, vortexed and centrifuged at 12,000 rpm for 1 minute, the supernatant was transferred to a new 15 ml centrifuge tube, 10 times the volume of PBbinding buffer was added and mixed, the supernatant was transferred to a MinElute purification column, centrifuged at 12,000 rpm for 1 minute, the liquid was discarded, 750 μl of Buffer PE was added, the tube was centrifuged at 12,000 rpm for 1 minute, the liquid was discarded and the tube was centrifuged again for 2 minutes. Finally, place the purification column in a new 1.5 ml centrifuge tube, add 35 μl ddH2O or TE, incubate at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 1 minute to obtain about 30 μl of purified DNA fragments.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for extracting metagenomic DNA from ancient dental calculus, aiming to provide an efficient and reliable DNA extraction method for studying the diversity and evolution of ancient microorganisms. The method includes steps such as sample pretreatment, DNA extraction, purification and quality inspection. By optimizing the extraction reagents and methods, it is possible to minimize exogenous contamination and improve DNA extraction efficiency while maintaining DNA integrity. This method is applicable to different types of ancient dental calculus samples, and high-quality metagenomic DNA can be obtained in a relatively short time, providing a solid foundation for subsequent genome sequencing and analysis.

[0015] High-quality metagenomic DNA can be extracted from ancient dental calculus in a relatively short period of time, with significantly improved extraction efficiency and significantly reduced contamination rate, making it suitable for all types of ancient dental calculus samples. This method not only provides a powerful tool for scientific research, but also lays the foundation for genome sequencing and functional analysis of ancient dental calculus. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the state of the sample after preliminary treatment in Example 1.

[0017] Figure 2 It is the electrophoresis diagram of the DNA extracted from Example 1.

[0018] Figure 3 is the DNA damage rate of the sample product in Example 1.

[0019] Figure 4 This is the state of the sample after preliminary treatment in Example 2.

[0020] Figure 5 This is the electrophoresis diagram of the DNA extracted from Example 2, which shows that the DNA fragments are well preserved after the extraction is completed.

[0021] Figure 6 It is the DNA damage rate of the sample product in Example 2, indicating that the DNA damage rate after the extraction is consistent with the age and is ancient DNA.

[0022] Figure 7 This is the state of the sample after preliminary treatment in Example 3 Figure 8 It is the electrophoresis diagram of the DNA extracted from Example 3.

[0023] Fig. 9 is the DNA damage rate of the sample product in Example 3. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further described below in conjunction with embodiments.

[0025] Embodiment 1: The DNA extraction method of dental calculus samples from a Han Dynasty tomb in Shandong Province includes the following steps: 1. Preliminary processing All necessary supplies were sterilized with ultraviolet light for more than 30 minutes; use a soft brush dipped in 75% ethanol to gently brush away the dust on the surface of the teeth, and leave them to dry naturally at room temperature. Spread a layer of clean aluminum foil on the table, use a disposable dental needle to scrape the dental plaque on the surface of the teeth, and let the dental plaque fall naturally on the aluminum foil. Transfer the dental plaque on the aluminum foil to a 2 ml centrifuge tube and calculate the weight of the dental plaque sample. Each tube of sample should be between 550 mg as much as possible; weigh the sample weights of 450 mg, 471 mg, 523 mg, and 577 mg.

[0026] 2. Sample lysis 1 ml of lysis buffer (Invitrogen, EDTA (0.5 M), AM9260G; Invitrogen, proteinase K solution, 4333793) was added to a 2 ml centrifuge tube containing dental calculus samples, and the dental calculus was broken with a sterilized plastic inoculation loop (Jiangsu Shitai, 82121-1009) at 60 rpm. The samples were placed in a heating and shaking dry bath (Thermo Scientific, 88871004) at 55°C and 1000 rpm for digestion overnight to ensure that the samples were suspended and mixed. After digestion overnight, the centrifuge tube was placed in a rotary mixer (Thermo Scientific, 88882015) and rotated and mixed at room temperature for 48 hours.

[0027] 3. DNA extraction The digested sample was centrifuged at 12,000 rpm for 1 minute (eppendorf, Centrifuge 5418 R), 950 μl of the supernatant was transferred to a new 2 ml centrifuge tube, 1 ml of phenol: chloroform: isopropanol (25:24:1) mixture (Shanghai Bioengineering) was added, vortexed and centrifuged at 12,000 rpm for 1 minute, the supernatant was transferred to a new 2 ml centrifuge tube, and this process was repeated twice. Add 1 ml of chloroform, vortex and mix, centrifuge at 12,000 rpm for 1 minute, transfer the supernatant to a new 15 ml centrifuge tube, add 10 times the volume of PB binding buffer (QIAGEN 28006X4) and mix, transfer to a MinElute purification column (QIAGEN 28006X4), centrifuge at 12,000 rpm for 1 minute, discard the liquid, add 750 µl Buffer PE (QIAGEN28006X4), centrifuge at 12,000 rpm for 1 minute, discard the liquid and centrifuge for another 2 minutes. Finally, place the purification column in a new 1.5 ml centrifuge tube, add 35 μl ddH2O, incubate at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 1 minute to obtain 35 µl of purified DNA fragments.

[0028] 4. Result verification.

[0029] A spectrophotometer (Thermo Scientific, NanoDrop™ 8000) was used to identify the purity and concentration of the sample products. Table 1 shows the purity and concentration of the extracted products. An A260 / A280 ratio between 1.8 and 2.0 indicates high DNA purity, while an A230 / A280 ratio above 2.0 indicates low protein and other contaminants.

[0030] Table 1 Purity and concentration of products extracted from dental calculus samples from a Han Dynasty tomb in Shandong Sample No. A260 / A280 A230 / A280 Concentration (μg / μl) 1 1.85 2.10 12.1 2 1.88 2.15 22.5 3 1.92 2.05 16.3 4 1.90 2.20 28.7 Figure 1 This is the state of the sample after preliminary treatment, which shows that it is clean and free of pollution, and the sample itself is not damaged. Then electrophoresis is performed to check the degradation of the sample. Figure 2 This is a gel image of DNA after extraction, showing that the DNA read length is relatively uniform, dragging is not serious, and there are no obvious large modern DNA fragments. Use pmdtool to determine whether it meets the characteristics of ancient DNA. Figure 3 The terminal damage rate for software identification is consistent with the era and there is no modern pollution.

[0031] Embodiment 2: The DNA extraction method of dental calculus samples from the wilderness site of the late Neolithic period in Guangxi is as follows: 1. Preliminary processing Due to the different soil and water conditions in Guangxi, this batch of samples has been severely corroded. All necessary supplies are sterilized with UV light for more than 30 minutes. Use a soft brush dipped in 75% ethanol to gently brush away the dust on the surface of the teeth, and let it dry naturally at room temperature. Scrape the dental calculus and calculate the weight of the dental calculus sample. Due to the different soil and water conditions in Guangxi, this batch of samples has been severely corroded. Therefore, the weights of the samples with less weight are 441mg, 320mg, 314mg, 338mg, and 290mg.

[0032] 2. Sample lysis 1 ml of lysis buffer (Invitrogen, EDTA (0.5 M), AM9260G; Invitrogen, proteinase K solution, 4333793) was added to a 2 ml centrifuge tube containing dental calculus samples, and the dental calculus was manually broken with a sterile plastic inoculation loop (Jiangsu Shitai, 82121-1009) at 60 rpm. The samples were placed in a heating and shaking dry bath (Thermo Scientific, 88871004) at 55°C and 1000 rpm for digestion overnight to ensure that the samples were suspended and mixed. After digestion overnight, the centrifuge tube was placed in a rotary mixer (Thermo Scientific, 88882015) and digested for 48 hours at room temperature.

[0033] 3. DNA extraction The digested sample was centrifuged at 12,000 rpm for 1 min (eppendorf, Centrifuge 5418 R), 900 μl of the supernatant was transferred to a new 2 ml centrifuge tube, 1 ml of phenol: chloroform: isopropanol (25:24:1) mixture (Shanghai Bioengineering) was added, vortexed and centrifuged at 12,000 rpm for 1 min, the supernatant was transferred to a new 2 ml centrifuge tube, and this process was repeated twice. Add 1 ml of chloroform, vortex and mix, centrifuge at 12,000 rpm for 1 minute, transfer the supernatant to a new 15 ml centrifuge tube, add 10 times the volume of PB binding buffer (QIAGEN 28006X4) and mix, transfer to a MinElute purification column (QIAGEN 28006X4), centrifuge at 12,000 rpm for 1 minute, discard the liquid, add 750 µl Buffer PE (QIAGEN28006X4), centrifuge at 12,000 rpm for 1 minute, discard the liquid and centrifuge for another 2 minutes. Repeat the previous step once. Then place the purification column in a new 1.5 ml centrifuge tube, add 35 μl ddH2O, incubate at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 1 minute to obtain 35 µl of purified DNA fragments.

[0034] 4. Result verification.

[0035] A spectrophotometer (Thermo Scientific, NanoDrop™ 8000) was used to identify the purity and concentration of the sample product. Table 2 shows the purity and concentration of the extracted product. A260 / A280 ratios between 1.8 and 2.0 indicate high DNA purity, while A230 / A280 ratios above 2.0 usually indicate low protein and other contaminants. Table 2 shows that the DNA purity is high after the extraction is completed.

[0036] Table 2 Purity and concentration of products extracted from dental calculus samples from a field site in the late Neolithic period in Guangxi Sample No. A260 / A280 A230 / A280 Concentration (μg / μl) 1 1.91 2.12 21.4 2 1.89 2.17 16.5 3 1.86 2.22 17.3 4 1.93 2.09 21.1 5 1.88 2.14 25.2 Figure 4 This is the state of the sample after preliminary treatment, which shows that it is clean and free of pollution, and the sample itself is not damaged. Then electrophoresis is performed to check the degradation of the sample. Figure 5 This is a gel image of DNA after extraction, showing that the DNA read length is relatively uniform, dragging is not serious, and there are no obvious large modern DNA fragments. Use pmdtool to determine whether it meets the characteristics of ancient DNA. Figure 6 The terminal damage rate for software identification is consistent with the era and there is no modern pollution.

[0037] Embodiment 3: The DNA extraction method of dental calculus samples from a Ming Dynasty tomb in Jiangsu Province comprises the following steps: 1. Preliminary processing All necessary supplies are sterilized with ultraviolet light for more than 30 minutes. Use a soft brush dipped in 75% ethanol to gently brush away the dust on the surface of the teeth, and let it dry naturally at room temperature. Spread a layer of clean aluminum foil on the table, use a disposable dental needle to scrape the tartar on the surface of the teeth, and let the tartar fall naturally on the aluminum foil. Transfer the tartar on the aluminum foil to a 2 ml centrifuge tube and calculate the weight of the tartar sample. Each tube of sample should be between 550 mg as much as possible. Weigh the sample weights to be 432 mg, 441 mg, 529 mg, and 660 mg.

[0038] 2. Sample lysis 1 ml of lysis buffer (Invitrogen, EDTA (0.5 M), AM9260G; Invitrogen, proteinase K solution, 4333793) was added to a 2 ml centrifuge tube containing dental calculus samples and broken with a sterile plastic inoculation loop (Jiangsu Shitai, 82121-1009) using an ultrasonic disruptor (Fisher Scientific 50) at 10 kHz until there was no obvious debris. The sample was placed in a heating and shaking dry bath (Thermo Scientific, 88871004) at 55°C and 1000 rpm for digestion overnight to ensure that the sample was suspended and mixed. After digestion overnight, the centrifuge tube was placed in a rotary mixer (Thermo Scientific, 88882015) and rotated and mixed at room temperature for 48 hours.

[0039] 3. DNA extraction The digested sample was centrifuged at 12,000 rpm for 1 minute (eppendorf, Centrifuge 5418 R), 950 μl of the supernatant was transferred to a new 2 ml centrifuge tube, 1 ml of phenol: chloroform: isopropanol (25:24:1) mixture (Shanghai Bioengineering) was added, vortexed and centrifuged at 12,000 rpm for 1 minute, the supernatant was transferred to a new 2 ml centrifuge tube, and this process was repeated twice. Add 1 ml of chloroform, vortex and mix, centrifuge at 12,000 rpm for 1 minute, transfer the supernatant to a new 15 ml centrifuge tube, add 10 times the volume of PB binding buffer (QIAGEN 28006X4) and mix, transfer to a MinElute purification column (QIAGEN 28006X4), centrifuge at 12,000 rpm for 1 minute, discard the liquid, add 750 µl Buffer PE (QIAGEN28006X4), centrifuge at 12,000 rpm for 1 minute, discard the liquid and centrifuge for another 2 minutes. Finally, place the purification column in a new 1.5 ml centrifuge tube, add 35 μl ddH2O, incubate at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 1 minute to obtain 35 µl of purified DNA fragments.

[0040] 4. Result verification.

[0041] A spectrophotometer (Thermo Scientific, NanoDrop™ 8000) was used to identify the purity and concentration of the sample products. Table 3 shows the purity and concentration of the extracted products. An A260 / A280 ratio between 1.8 and 2.0 indicates high DNA purity, while an A230 / A280 ratio above 2.0 indicates low protein and other contaminants.

[0042] Table 3 Purity and concentration of products extracted from dental calculus samples from ancient tombs in Jiangsu Sample No. A260 / A280 A230 / A280 Concentration (μg / μl) 1 1.62 1.88 9.3 2 1.26 1.32 12.8 3 1.12 1.67 11.5 4 1.46 1.78 4.4 Figure 7 This is the state of the sample after preliminary treatment, which shows that it is clean and free of pollution, and the sample itself is not damaged. Then electrophoresis is performed to check the degradation of the sample. Figure 8 This is a gel image of DNA after extraction, showing that the DNA read length is not uniform, the dragging is serious, and the fragment length is mostly less than 50bp. Use pmdtool to determine whether it meets the characteristics of ancient DNA. Fig. 9 The software identified that the terminal damage rate was consistent with the age and there was no modern pollution, but the fragments were short and the quality was low, resulting in an unsmooth curve and low quality. By comparing the results of Examples 1 and 3, it can be seen that low-frequency fragmentation plays a crucial role in the extraction of dental calculus DNA.

[0043] Finally, although this specification is described according to implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A method for extracting metagenomic DNA from ancient dental calculus, characterized in that: The following steps are involved: (1) Sample pretreatment: Use ethanol to clean the tooth surface and scrape off the dental calculus on the tooth surface; (2) Sample lysis: Add lysis solution to the dental calculus sample and then perform low-frequency crushing; (3) Sample digestion; (4) DNA extraction.

2. The method for extracting metagenomic DNA from ancient dental calculus according to claim 1, characterized in that: In the step (1), the ethanol concentration used is 75%, and all the instruments and equipment used are sterilized by ultraviolet light.

3. The method for extracting metagenomic DNA from ancient dental calculus according to claim 1, characterized in that: In the step (2), the lysis solution is prepared by adding 10 μl of 20 mg / ml proteinase K to 990 μl of 0.5 M EDTA solution; the solution is crushed at a low frequency of 60 rpm until no debris is visible to the naked eye.

4. The method for extracting metagenomic DNA from ancient dental calculus according to claim 1, characterized in that: In step (3), the sample is placed in a metal bath with a shaking function, and digested overnight at 55°C and 1000 rpm to ensure that the sample is suspended and mixed; after digesting overnight, the sample is placed in a rotary mixer and digested for 48 hours at room temperature.

5. The method for extracting metagenomic DNA from ancient dental calculus according to claim 1, characterized in that: In step (4), the digested sample was centrifuged at 12,000 rpm for 1 minute, 950 μl of the supernatant was aspirated into a new 2 ml centrifuge tube, 1 ml of a mixture of phenol:chloroform:isopropanol (25:24:1) was added, vortexed and centrifuged at 12,000 rpm for 1 minute, and the supernatant was transferred to a new 2 ml centrifuge tube; 1 ml of chloroform was added, vortexed and centrifuged at 12,000 rpm for 1 minute, the supernatant was transferred to a new 15 ml centrifuge tube, 10 times the volume of PB binding buffer was added and mixed, the supernatant was transferred to a MinElute purification column, centrifuged at 12,000 rpm for 1 minute, the liquid was discarded, 750 μl of Buffer PE was added, the column was centrifuged at 12,000 rpm for 1 minute, the liquid was discarded and centrifuged for another 2 minutes; finally, the purification column was placed in a new 1.5 ml centrifuge tube, 35 μl of ddH2O or TE was added, incubated at room temperature for 2 minutes, and then centrifuged at 12,000 rpm. Centrifuge at rpm for 1 minute to obtain approximately 30 µl of purified DNA fragments.

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