Method for reducing exogenous DNA pollution in keratinized DNA extract
Through the combination of enzymatic lysis and alkali removal treatment, the problem of exogenous DNA contamination in keratinized DNA extracts is solved, the stability and enrichment efficiency of DNA is improved, and it is suitable for various keratinized tissues, reducing costs and improving Accuracy of experimental results.
Patent Information
- Application Number
- CN202411437636.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-15
- Publication Date
- 2024-12-06
AI Technical Summary
In wildlife studies, exogenous DNA contamination often occurs in keratinized DNA extracts, resulting in loss of typing sites, low stability and false results.
Different types of keratinized tissues were cleaned by combining enzymatic treatment (EnzW) and alkaline treatment (AlkW), and the attached exogenous contaminants were decomposed by using protease (PK) and sulfuric acid alcohol (SDS), and the attached exogenous contaminants were decomposed by strong Alkali and EDTA destroy cell membranes and remove bacterial DNA contamination.
It effectively reduces exogenous DNA contamination in keratinized DNA extracts, improves DNA stability and endogenous DNA enrichment efficiency, reduces experimental costs, and is suitable for various keratinized tissues.
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Figure CN119082102A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a method for reducing exogenous DNA contamination in keratinized DNA extracts. Background Art
[0002] In the field of wildlife research, keratinized tissue is a common, easy-to-collect, easy-to-store non-destructive animal sample, mainly including the animal's epidermis and its attachments, such as horns, hooves, snake sloughs, hair, scales, thorns and feathers. Keratinized tissue is not only a strong external protective barrier for the animal body, protecting it from bacteria, viruses and other microorganisms, but also plays an important role in the animal's eating, attacking or defending behaviors. Like other animal tissues, keratinized tissue also contains a certain quality of genomic DNA (referred to as: keratinized DNA). Studies have shown that the extraction results based on keratinized DNA are often unsatisfactory, specifically manifested in the loss of typing sites, low stability, and even erroneous results. Researchers have found that the above situation mostly occurs in experiments related to nuclear genes (nuDNA). Further studies have shown that the fragmentation level of nuDNA in keratinized DNA is significantly higher than that of mitochondrial DNA (mtDNA), and the excessive proportion of exogenous DNA contamination (mainly bacterial DNA) in keratinized DNA extracts is also the main factor leading to "poor" experimental results. In addition, keratinized DNA has the characteristics of fewer DNA fragments and more fragmented DNA fragments.
[0003] In order to solve the problems of loss of typing sites, low stability and erroneous results in the process of keratinized tissue DNA extraction in the prior art, those skilled in the art are eager to develop a method for effectively reducing exogenous DNA contamination in keratinized DNA extracts. Summary of the invention
[0004] The present invention aims to solve the problems of loss of typing sites, low stability and erroneous results in the process of extracting DNA from keratinized tissue in the prior art, and provides a method for effectively reducing exogenous DNA contamination in keratinized DNA extracts.
[0005] One of the purposes of the present invention is to provide a method for reducing exogenous DNA contamination in keratinized DNA extracts, the method comprising the following steps:
[0006] S1: Hair and barb samples: Use sterile surgical scissors to cut hair or barb tissue 0.5-1 cm away from the animal epidermis, weigh 1-10 mg of tissue sample according to experimental requirements, and perform DetW, EnzW and AlkW cleaning in turn;
[0007] S2: Horn and hoof samples: Use a hard brush to clean the horn or hoof samples in 0.1% laundry detergent, rinse with sterile water three times after 30 seconds, grind with a 6.5mm twist drill, and clean the surface debris. Finally, take 10-20mg of the above-treated samples and place them in a centrifuge tube for EnzW cleaning.
[0008] S3: The outer keratin part of the tortoise shell and the feather shaft sample: Use a brush to clean it in 0.1% laundry detergent, rinse it with sterile water 3 times after 30 seconds, use scissors to remove the root, and take 10-20 mg of the above-treated sample from the near root end upwards, put it into a centrifuge tube, cut it into pieces, and perform EnzW cleaning treatment.
[0009] In a preferred embodiment of the present invention, the DetW cleaning treatment step in S1 is: put the keratinized sample to be tested into a culture dish filled with 0.1% laundry detergent solution, rub it by hand for 30 seconds, discard the waste liquid, rinse it with sterile water for 3 times, and then transfer it to a sterile centrifuge tube.
[0010] In a preferred embodiment of the present invention, the EnzW cleaning treatment step in S1 is: place the keratinized sample to be tested in a centrifuge tube, add 15 μL PK, 35 μL SDS and 350 μL TNE buffer to the centrifuge tube, then place it in a 56°C water bath and heat it for 2 hours, discard the waste liquid, and then rinse it three times with 1 mL of sterile water.
[0011] In a preferred embodiment of the present invention, the PK concentration is 25 mg / mL,
[0012] In a preferred embodiment of the present invention, the SDS concentration is 10%.
[0013] In a preferred embodiment of the present invention, the composition of the TNE buffer is: Tris solution-10 mM, NaCl-100 mM, EDTA-2.5 mM, and the pH of the TNE buffer is 7.6.
[0014] In a preferred embodiment of the present invention, the AlkW cleaning treatment step in S1 is: placing the keratinized sample to be tested into a centrifuge tube, adding a mixed solution containing NaOH and EDTA to the centrifuge tube, then heating it in a water bath at 56°C for 2.5min-3.5min, discarding the waste liquid, and repeatedly rinsing with sterile water 4 times.
[0015] In a preferred embodiment of the present invention, the concentration of NaOH in the mixed solution is 0.2 M, and the concentration of EDTA in the mixed solution is 2.5 mM.
[0016] Beneficial effects of the present invention: The present invention provides a method for reducing exogenous DNA contamination in keratinized DNA extracts, and the method can adopt different treatment schemes for different types of keratinized tissues, including: hair and barbs, horns and hooves, the outer keratin part of tortoise shells and keratinized tissues of feather shafts; the present invention removes exogenous DNA contamination by enzymatic hydrolysis (EnzW) of hard keratinized tissues such as horns and hooves, and further removes bacterial DNA contamination by adding alkaline removal treatment (AlkW) to soft keratinized tissues such as hair and barbs on the basis of enzymatic hydrolysis (EnzW).
[0017] In the EnzW treatment, PK and SDS are used to effectively decompose exogenous pollutants attached to the surface of keratinized tissue and release DNA, while the keratinized DNA is wrapped in high-sulfur keratin bundles and will not precipitate, thereby effectively reducing exogenous DNA contamination in keratinized DNA extracts.
[0018] In the AlkW treatment, the corrosiveness of strong alkali at a certain temperature is used to quickly destroy the cell membrane and nuclear membrane, further removing the contamination of bacterial DNA. In the process of alkali solution treatment, special emphasis is placed on the precise control of the treatment time. The sample is heated in a water bath at 56°C for 2.5min-3.5min, and the longest time is no more than 4 minutes, so as to avoid the destruction of the sample structure to the greatest extent, thereby ensuring the stability of the digestion effect; at the same time, 2.5mM EDTA is used to recognize and act on the lipopolysaccharide structure on the cell membrane surface of Gram-negative bacteria, causing the structure to disintegrate and the cell membrane to rupture, which helps other antibacterial agents penetrate into the cell, thereby reducing bacterial DNA contamination.
[0019] The present invention provides a method for reducing exogenous DNA contamination in keratinized DNA extracts, effectively reducing the proportion of bacterial DNA and ensuring the enrichment efficiency of endogenous DNA; compared with common ultrasonic and ultraviolet decontamination methods, the treatment process of the method is common reagents and equipment in molecular laboratories, which reduces costs and improves efficiency, and has the advantages of low cost and simple operation. The method for reducing exogenous DNA contamination in keratinized DNA extracts provided by the present invention is suitable for all types of keratinized tissues, and improves the applicability of various keratinized tissues. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 Primer N p 、N m1 and N m2 Melting curve diagram of
[0021] Figure 2 The figures are comparison results of Ct values of mtDNA and pDNA in different types of keratinized samples; A and B are comparisons of Ct values of intact hard keratinized tissue before and after treatment, and C and D are comparisons of Ct values of damaged hard keratinized tissue or soft keratinized tissue before and after treatment. DETAILED DESCRIPTION
[0022] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.
[0023] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments and the accompanying drawings of the specification. The test methods used in the following examples are conventional methods unless otherwise specified, and the materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained by those skilled in the art through commercial channels.
[0024] Embodiment 1:
[0025] S1: Hair and barb samples: Use sterile surgical scissors to cut hair or barb tissue 0.5-1 cm away from the animal epidermis, weigh 1-10 mg of tissue sample according to experimental requirements, and perform DetW, EnzW and AlkW cleaning in turn;
[0026] S2: Horn and hoof samples: Use a hard brush to clean the horn or hoof samples in 0.1% laundry detergent, rinse with sterile water three times after 30 seconds, grind with a 6.5mm twist drill, and clean the surface debris. Finally, take 10-20mg of the above-treated samples and place them in a centrifuge tube for EnzW cleaning.
[0027] S3: The outer keratin part of the tortoise shell and the feather shaft samples were cleaned with a brush in 0.1% laundry detergent, rinsed with sterile water for 30 seconds, the roots were removed with scissors, and 10-20 mg of the above-treated samples were taken from the near-root end and placed in a centrifuge tube, cut into pieces, and then cleaned with EnzW.
[0028] S4: Obtain the tissue samples to be tested after treatment in the experimental group (denoted as: NE, N is the species ID);
[0029] The DetW cleaning treatment steps are as follows: placing the keratinized sample to be tested in a culture dish containing a 0.1% laundry detergent solution, rubbing it by hand for 30 seconds, discarding the waste liquid, rinsing it with sterile water for 3 times, and then transferring it to a sterile centrifuge tube;
[0030] The EnzW cleaning treatment steps are as follows: placing the keratinized sample to be tested in a centrifuge tube, adding 15 μL PK, 35 μL SDS and 350 μL TNE buffer (Tris solution-10 mM, NaCl-100 mM, EDTA-2.5 mM, pH of the TNE buffer = 7.6) to the centrifuge tube, then heating in a 56°C water bath for 2 hours, discarding the waste liquid, and then washing with 1 mL of sterile water for 3 times; the PK concentration is 25 mg / mL, and the SDS concentration is 10%;
[0031] The AlkW cleaning treatment steps are as follows: placing the keratinized sample to be tested into a centrifuge tube, adding a mixed solution containing NaOH and EDTA (the NaOH concentration in the mixed solution is 0.2M, and the EDTA concentration is 2.5mM) into the centrifuge tube, then heating in a water bath at 56°C for 2.5min-3.5min, discarding the waste liquid, and repeatedly rinsing with sterile water 4 times.
[0032] Comparative Example 1:
[0033] Use sterile surgical scissors to cut the tissue to be tested at 0.5-1 cm away from the animal epidermis as a sample. The information of different types of samples to be tested is shown in Table 1. According to the experimental requirements, 1-10 mg of the above-mentioned tissue samples to be tested are weighed, and the above-mentioned samples to be tested are subjected to DetW cleaning treatment. The DetW cleaning treatment steps are: put the keratinized sample to be tested into a culture dish filled with 0.1% laundry detergent solution, rub it with hands for 30 seconds, discard the waste liquid, rinse it with sterile water 3 times, and then transfer it to a sterile centrifuge tube to obtain the tissue sample to be tested after the control group treatment (recorded as: NR, N is the species ID).
[0034] Effect experiment:
[0035] (1) The treated tissue samples obtained in Example 1 and the comparative example were added to the digestion buffer A (350 μL, 50 mM-Tris-base, 100 mM-NaCl, 10 mM-Ca 2+ , pH = 7.6), PK (30 μL, 25 mg / mL), DTT (100 μL, 1 M) and SDS (60, 10%) for digestion, followed by enrichment using a magnetic bead kit (Animal and Plant DNA Extraction Kit, Beijing Zhongke Leiming Co., Ltd.), for specific steps see the instruction manual, and then the concentration of the extracted DNA stock solution was detected using a qubit 4.0 quantitative analyzer.
[0036] The quantitative results of the 12 keratinized tissue samples are shown in Table 1. Taking the control group (NR) as an example, the amount of genomic DNA enriched from different types of keratinized samples varied greatly, ranging from 6.83 ng / mg to 13.42 ng / mg, with an average of 9.71 ± 3.16 ng / mg. However, the amount of DNA in each experimental group after treatment with EnzW and AlkW decreased, with an average of 3.88 ± 2.70 ng / mg (1.02-8.85 ng / mg).
[0037] Table 1
[0038]
[0039] In this example, PCR amplification was performed on the DNA of the samples to be tested (experimental group and control group) obtained in step (1). The PCR amplification system was 10 μL, including 1 μL DNA solution, 5 μL TB Green Premix ExTaqII (Takara, Dalian), 0.4 μL of each of the upstream and downstream primers (10 μmol / L), and 6 ng of DNA stock solution, which was filled up to 10 uL with sterile water. Primer N was used for the detection of DNA samples of rachis and barbs. m1 The nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.2. The other four tissue samples DNA were detected using primers N m2 The nucleotide sequences are shown in SEQ ID NO.3 and SEQ ID NO.4. The amplification primers for bacterial DNA are p The nucleotide sequences are shown in SEQ ID NO.5 and SEQ ID NO.6. The PCR amplification conditions were 95°C pre-denaturation for 30s; 95°C denaturation for 5s, 60°C annealing for 34s, 72°C extension for 30s, 40 cycles; extension at 72°C for 10s after the cycle; fluorescence quantitative PCR was performed using a CFX384 Real-Time System fluorescence quantitative instrument (BIO-RAD, USA), and the fluorescence signal was immediately collected and the results were displayed after each cycle extension step.
[0040] The Ct value (Ct mt ) detection, and calculate the difference; the bacterial pDNA of the same tissue sample to be tested in Example 1 and Comparative Example 1 is tested for Ct value (Ct p) detection, and calculate the difference; in this embodiment, the ratio of the above difference and the change of the Ct value are used as the standard for measuring the bacterial removal effect. When the ratio of the above difference is lower, the more the Ct value of pDNA increases, it means that the number of bacterial pDNA copies in the sample DNA to be tested is smaller, indicating that the treatment effect of bacterial DNA contamination in the sample DNA to be tested is better.
[0041] Primer N for amplification of bacterial DNA p The detection of DNA in the tissue sample to be tested uses primer N m1 and N m2 The melting curve of the fluorescent PCR amplification changes as follows Figure 1 As shown in the figure, it was found that the melting curves of the above two primers had only one significant peak, and the peak values were around 81°C, 86°C and 84°C, respectively, indicating that the above N p 、N m1 and N m2 The quality of the primers was good, with no nonspecific amplification and dimers, meeting the requirements of the experiment.
[0042] For intact thick keratinized tissues (horns, hooves, and feather rachis), the Ct values of mtDNA in the control group (Ct mt )like Figure 2 As shown in A, the Ct values of the three control groups mt The average value was 22.39±1.82, and the Ct mt (24.57±1.88) was 2.18±0.13 ( Figure 2 B). The Ct value of bacterial DNA in the experimental group (24.05±2.57) was only 1.28±0.22 higher than that in the control group (25.33±2.66). This indicates that this type of tissue contains less bacterial DNA and can be washed routinely if well preserved. Additional washing may result in a decrease in the proportion of host DNA in the DNA extract.
[0043] For damaged hard keratinized tissue (external surface of turtle shell) or soft keratinized tissue (hair and barbs), the Ct value of mtDNA in the control group (Ct mt )like Figure 2 As shown in C, the Ct values of the three control groups mt The average value was 25.45±1.34, and the Ct mt (27.5±0.41) was 2.05±4.30 higher than the control group. However, the Ct value of bacterial DNA in the experimental group (24.17±5.19) was significantly higher than that in the control group (19.87±4.49) by 4.29±0.93 ( Figure 2 D) This indicates that this type of tissue contains more bacterial DNA and that additional washing can significantly increase the proportion of host DNA.
[0044] In the above fluorescence quantitative experiment, the Ct value is negatively correlated with the copy number, indicating that the method provided by the present invention for reducing exogenous DNA contamination in keratinized DNA extracts, especially the damaged hard keratinized tissue and soft keratinized tissue, has a significantly higher reduction in the amount of bacterial DNA contamination in the DNA extracted from the tested tissue samples than that of mtDNA, indicating that the method provided by the present invention can significantly remove the contamination of exogenous DNA while ensuring the enrichment efficiency of endogenous DNA.
[0045] The contents not described in detail in the specification of the present invention are well-known technologies for those skilled in the art. Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be based on the definition of the claims.
Claims
1. A method for reducing exogenous DNA contamination in keratinized DNA extracts, characterized in that: The method comprises the following steps: S1: Hair and barb samples: Use sterile surgical scissors to cut hair or barb tissue 0.5-1 cm away from the animal epidermis, weigh 1-10 mg of tissue sample according to experimental requirements, and perform DetW, EnzW and AlkW cleaning in turn; S2: Horn and hoof samples: Use a hard brush to clean the horn or hoof samples in 0.1% laundry detergent, rinse with sterile water three times after 30 seconds, grind with a 6.5mm twist drill, and clean the surface debris. Finally, take 10-20mg of the above-treated samples and place them in a centrifuge tube for EnzW cleaning. S3: The outer keratin part of the tortoise shell and the feather shaft sample: Use a brush to clean it in 0.1% laundry detergent, rinse it with sterile water 3 times after 30 seconds, use scissors to remove the root, and take 10-20 mg of the above-treated sample from the near root end upwards, put it into a centrifuge tube, cut it into pieces, and perform EnzW cleaning treatment.
2. The method according to claim 1, characterized in that The DetW cleaning treatment steps described in S1 are: put the keratinized sample to be tested into a culture dish filled with 0.1% laundry detergent solution, rub it by hand for 30 seconds, discard the waste liquid, rinse it with sterile water for 3 times, and then transfer it to a sterile centrifuge tube.
3. The method according to claim 1, characterized in that The EnzW cleaning treatment steps described in S1 are: put the keratinized sample to be tested into a centrifuge tube, add 15 μL PK, 35 μL SDS and 350 μL TNE buffer into the centrifuge tube, then heat it in a 56°C water bath for 2 hours, discard the waste liquid, and then rinse it three times with 1 mL of sterile water.
4. The method according to claim 3, characterized in that: The PK concentration was 25 mg / mL.
5. The method according to claim 3, characterized in that: The SDS concentration was 10%.
6. The method according to claim 3, characterized in that The composition of the TNE buffer solution is: Tris solution-10 mM, NaCl-100 mM, EDTA-2.5 mM, and the pH of the TNE buffer solution is 7.
6.
7. The method according to claim 1, characterized in that The AlkW cleaning treatment steps described in S1 are: placing the keratinized sample to be tested into a centrifuge tube, adding a mixed solution containing NaOH and EDTA into the centrifuge tube, then heating it in a water bath at 56°C for 2.5min-3.5min, discarding the waste liquid, and repeatedly rinsing with sterile water for 4 times.
8. The method according to claim 7, characterized in that The concentration of NaOH in the mixed solution is 0.2 M, and the concentration of EDTA in the mixed solution is 2.5 mM.