Efficient extraction method of blue fox sperm DNA

Abstract

The invention discloses an efficient extraction method of blue fox sperm DNA, and belongs to the technical field of molecular biology. Aiming at the structural characteristics that chromatin of the blue fox sperms is highly condensed, histone is replaced by protamine, and a stable nucleoprotein complex is formed through an intermolecular disulfide bond, the invention provides an efficient DNA extraction method suitable for the blue fox sperms. According to the method, based on the synergistic effect of three chemical reagents, namely SDS, PK and DTT, full lysis of the sperm cells of the blue foxes is achieved, and then efficient enrichment of sperm DNA of the blue foxes can be achieved by combining a conventional DNA extraction method. According to the efficient extraction method of the blue fox sperm DNA provided by the invention, the yield and integrity of the blue fox sperm DNA are remarkably improved, and the limitation that sperm chromatin is difficult to effectively lyse by a conventional method is overcome; the method provides reliable technical support for research on genetic diversity evaluation, population management, molecular breeding and the like of the blue foxes, and has the potential of popularization and application in other high-condensation sperm species.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular biology, and particularly relates to a high-efficiency extraction method of blue fox sperm DNA. BACKGROUND

[0002] Blue fox (Vulpes lagopus) Vulpes lagopus ), also known as Arctic fox, white fox and snow fox, belongs to the order Carnivora and family Canidae. Due to its soft fur and good warmth retention, the blue fox has long occupied an important economic position in the global fur trade. Wild blue foxes are widely distributed in the Arctic and subarctic regions and are key predators in the local ecosystem, playing an important role in maintaining the stability of the food web and protecting biodiversity. Efficient acquisition and utilization of blue fox sperm DNA not only is a key measure to improve the level of reproductive management and promote genetic improvement, but also provides solid technical support for genetic monitoring of wild populations, thereby playing an irreplaceable role in the sustainable use and long-term protection of the species.

[0003] Sperm DNA and somatic cell DNA differ significantly in structure, mainly in the compression mode of the higher-order structure of chromatin and the protein component: in sperm, most histones are replaced by protamines, and DNA is covalently cross-linked with protamines through disulfide bonds to form a very dense and stable three-dimensional network structure, which makes the compression degree of chromatin about 6-10 times higher than that of somatic cells. This highly compressed structure not only effectively protects genetic material from damage before fertilization, but also significantly hinders the accessibility of external chemical reagents and enzymes to DNA, making subsequent lysis and extraction operations much more difficult.

[0004] Currently, most methods widely used for animal sperm DNA extraction are derived from or improved from somatic cell DNA extraction techniques, mainly including phenol-chloroform extraction, silica gel column method, magnetic bead method, etc. These methods meet the needs of sperm DNA extraction to some extent, but the lysis buffer system is not optimized for the highly dense chromatin structure and specific protein packaging form of sperm cells, often leading to insufficient lysis of sperm cells, limited DNA release efficiency, and thus low total yield, which seriously affects the sensitivity and accuracy of downstream applications. To overcome the above limitations, researchers have continuously optimized the composition of lysis buffer, introduced specific enzyme digestion steps (such as micrococcal nuclease digestion), and used specially designed commercial kits (such as SpermX ™ extraction system, QIAamp DNA Investigator kit, etc.) to improve sperm DNA extraction efficiency. However, such methods are usually applied to human forensics, and there are still certain limitations in cost, operating conditions, cross-species applicability, etc., which need to be further optimized and verified to meet the needs of different research and application scenarios.

[0005] Due to the significant structural differences between sperm of different species, the DNA extraction method needs to be accurately adapted according to its unique morphological characteristics and biochemical properties. Currently, research in this field is mostly focused on humans and conventional livestock models (such as cattle, sheep, pigs, and rodents), while there is still a significant gap in the research on the special structure and adaptive extraction method of blue fox sperm. Therefore, developing a targeted DNA extraction method based on the structural characteristics of blue fox sperm, which has the advantages of operational safety, economy, simplicity, and high efficiency of lysis, has become a technical demand that needs to be addressed in this field. SUMMARY

[0006] The present application aims to solve the problem that the DNA extraction efficiency is low and the quality is unstable due to the difficulty in effectively lysing the blue fox sperm due to its unique structural specificity, and provides a high-efficiency extraction method for blue fox sperm DNA.

[0007] One of the purposes of the present application is to provide a high-efficiency extraction method for blue fox sperm DNA, which comprises the following steps: S1: mixing blue fox semen and TNE buffer according to a volume ratio of 1:1, then adding them into a DNA centrifuge tube to obtain a mixed solution; S2: sequentially adding SDS, PK and DTT solutions to the mixed solution obtained in S1, oscillating and mixing, and performing digestion treatment; S3: performing DNA extraction on the mixed solution after digestion treatment in S2 to obtain blue fox sperm DNA.

[0008] Preferably, the total number of sperm in the blue fox semen in S1 is 2×10 7 -2×10 6 .

[0009] Preferably, the total number of sperm in the blue fox semen is 1×10 7 .

[0010] Preferably, the concentration of SDS in S2 is 15% (w / v), and the addition amount is 40 μL.

[0011] Preferably, the concentration of PK in S2 is 20 mg / mL, and the addition amount is 20 μL.

[0012] Preferably, the concentration of DTT in S2 is 1.5 mol / L, and the addition amount is 20 μL.

[0013] Preferably, the temperature of the digestion treatment in S2 is 56℃, and the reaction time is 2 h.

[0014] Preferably, the method of DNA extraction in S3 includes but is not limited to phenol-chloroform-isoamyl alcohol extraction method, silica gel column method and magnetic bead method.

[0015] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a highly efficient method for extracting DNA from blue fox sperm. By synergistically optimizing the concentration combination of three key lysis reagents—sodium dodecyl sulfate (SDS), dithiothreitol (DTT), and proteinase K (PK)—a highly efficient DNA lysis system suitable for blue fox sperm was established. Furthermore, by limiting the number of blue fox sperm (range 2 × 10⁻⁶)... 6 -2×10 7 The optimal value is 1×10 7 By using the following digestion conditions (56℃, 2 h), optimal lysis conditions (40 μL 15% SDS, 20 μL 20 mg / mL PK and 20 μL 1.5 mol / L DTT), combined with conventional DNA extraction methods, high concentrations and well-preserved blue fox sperm DNA can be obtained.

[0016] The method has the following advantages: (1) High safety: The use of water-soluble reagents SDS, DTT and PK results in low experimental risk and is suitable for routine laboratory conditions; (2) High convenience: Only specific concentrations of reagents need to be added to complete the lysis, the process is simple and time-saving, and it is suitable for high-throughput sample processing; (3) Good economy: The reagents used are all routinely available and inexpensive, which is convenient for large-scale experimental promotion; (4) Easy to kitteria: The method has standardized steps and clear components, and can be further developed into a ready-to-use detection kit to improve the standardization and convenience of experimental operations.

[0017] The efficient method for extracting sperm DNA from blue foxes provided by this invention, although specifically developed for the sperm structure of the blue fox (a non-model animal), is applicable to other mammals as well, particularly sperm cells with highly condensed chromatin. It possesses good versatility and potential for wider application, and can be further extended to fields such as wildlife germplasm resource preservation, phylogenetic analysis, and research on reproductive-related molecular mechanisms, providing stable and reliable technical support for related research. This invention not only represents a core pathway to overcome the bottlenecks of existing methods but also provides crucial technical assurance for molecular breeding and population genetic diversity research. Attached Figure Description

[0018] Figure 1 A three-dimensional scatter plot showing the relationship between the concentration of extracted blue fox sperm DNA and the concentrations of lysis reagents SDS, PK, and DTT. Figure 2 Electrophoresis image of blue fox sperm DNA extracted by silica gel column method under optimal lysis conditions; Figure 3The electrophoretogram of the sperm DNA of the blue fox extracted by the phenol-chloroform-isoamyl alcohol extraction method under the optimal lysis condition; Figure 4 The electrophoretogram of the sperm DNA of the blue fox extracted by the magnetic bead method under the optimal lysis condition. DETAILED DESCRIPTION

[0019] Those skilled in the art can improve the process parameters according to the content herein. It is particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by the preferred embodiments, and the related personnel can obviously modify or appropriately change and combine the method and application described herein without departing from the content and scope of the present application, to realize and apply the present application technology.

[0020] In order to make the purpose, technical solutions and advantages of the present application more clear and obvious, the present application will be further described in detail below in combination with specific embodiments. The experimental methods used in the following examples are conventional methods unless otherwise specified. 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 commercial channels by those skilled in the art.

[0021] Example 1: Collection of blue fox sperm samples The sperm samples were collected from 6 adult healthy male blue foxes (2-3 years old, 4.5-6.0 kg in weight), which were confirmed to have no reproductive system diseases by clinical examination. The semen was collected by rectal electrical stimulation method (voltage 5-8 V, pulse frequency 30 Hz, stimulation duration 3-5 seconds / time). In order to ensure the comparability of the experimental results, the samples with sperm density ≥5×10 8 / mL were included after evaluation by a computer-aided semen analysis system immediately after collection, centrifuged, and the above sperm samples were diluted to 5×10 6 / mL, 1×10 7 / mL, 5×10 7 / mL, 1×10 8 / mL, 5×10 8 / mL, respectively numbered as H1-H6, and stored at 4℃ for a short period (≤5 days).

[0022] Example 2: Extraction of blue fox sperm DNA by silica gel column method Take 200 μL of the blue fox semen samples (sperm density 5×10 7 / mL) were mixed with equal volume of 200 μL TNE buffer (1x, pH 7.4) respectively, and then 40 μL SDS (5%, 10%, 15%, 20% w / v), 20 μL PK (10, 15, 20, 25 mg / mL) and 20 μL DTT solution (0.5, 1.0, 1.5, 2.0 mol / L) of different concentrations were added in turn, and the mixture was vortexed for 30 s to mix well, and then was placed in a 56°C constant temperature metal heater for digestion for 2 h, and was gently inverted to mix every 15 min during the digestion; the mixture after digestion was subjected to DNA extraction using a UE multi-source genomic DNA small amount extraction kit (purchased from USEVERBRIGHT, USA; the kit is a silica gel column method kit, containing DNA preparation tube, Buffer C-L, Buffer P-D, Buffer W1, Buffer W2 and Eluent).

[0023] The steps for extracting blue fox sperm DNA using the kit are as follows: S1: The mixture after digestion was mixed with 350 μL Buffer P-D in a centrifuge tube, and was mixed well by vortexing, and then was centrifuged at 12000 rpm for 10 min at room temperature, and the precipitate was discarded to obtain the supernatant; S2: The DNA preparation tube was placed in a 2 mL centrifuge tube, and the supernatant obtained in S1 was transferred to the DNA preparation tube, and was centrifuged at 12000 rpm for 1 min; the filtrate was removed, and the DNA preparation tube was transferred to a new 2 mL centrifuge tube, and then 500 μL Buffer W1 was added, and was centrifuged at 12000 rpm for 1 min at room temperature; S3: The filtrate after centrifugation in S2 was removed, and the DNA preparation tube was transferred to a new 2 mL centrifuge tube, and then 700 μL Buffer W2 was added, and was centrifuged at 12000 rpm for 1 min at room temperature; this step was repeated twice; S4: The filtrate after centrifugation in S3 was removed, and the DNA preparation tube was transferred to a new 2 mL centrifuge tube, and was centrifuged at 12000 rpm for 1 min; the DNA preparation tube was transferred to a new 1.5 mL centrifuge tube, and 100 μL Eluent (which needs to be preheated to 65°C before use) was added to the center of the membrane of the preparation tube, and was placed at room temperature for 3 min, and then was centrifuged at 12000 rpm for 2 min to elute, and the DNA was obtained.

[0024] Effect experiment: 1. DNA concentration detection The blue fox sperm DNA obtained under different lysis conditions was subjected to concentration determination using an ultramicro spectrophotometer, and the specific steps were as follows: First, the ultramicro spectrophotometer self-checking is carried out, and preheating for 2-3 min; initialize the software, select "nucleic acid (DNA / RNA)" measurement mode; use the same solvent as the DNA to be measured (Eluent is selected in this experiment), take 1 μL in the detection area, touch the center of the area, avoid bubbles; close the measurement arm, click "blank" calibration, ensure that the baseline absorbance is zero; use a dust-free paper to wipe the detection area clean, take 1 μL of the DNA to be measured and drop it into the detection area; close the measurement arm, click "sample" to measure the DNA concentration (unit: ng / µL); after the measurement is completed, use a dust-free paper to wipe the detection area with ultrapure water, then use a dry dust-free paper to wipe it dry to avoid residue. To ensure data reliability, the DNA extracted from blue fox sperm under different lysis conditions is independently repeated 3 times.

[0025] The DNA concentration data of blue fox sperm samples numbered H1-H6 extracted under different lysis conditions is shown in Table 1.

[0026] Among them, the DNA extraction concentration range of sample H1 is 2.983±0.409-41.950±1.558 ng / µL, the average value is 18.488±12.208 ng / µL; sample H2 is 2.980±0.184-43.700±1.952 ng / µL, the average value is 18.432±12.251 ng / µL; sample H3 is 2.933±0.249-41.767±1.519 ng / µL, the average value is 18.404±12.167 ng / µL; sample H4 is 3.017±0.409-44.100±3.310 ng / µL, the average value is 18.423±12.216 ng / µL; sample H5 is 2.983±0.189-42.967±1.551 ng / µL, the average value is 18.342±12.085 ng / µL; sample H6 is 3.050±0.455-43.033±1.723 ng / µL, the average value is 18.368±12.122 ng / µL; the DNA concentration range and average value of blue fox sperm samples numbered H1-H6 extracted under different lysis conditions are consistent p >0.05).

[0027] Therefore, under the condition of inputting the same number (and the same sperm density) of sperm samples, the efficient blue fox sperm DNA extraction method provided by this invention exhibits good reproducibility. The differences in the concentration of the extracted DNA are mainly due to different lysis conditions, rather than individual differences. It is worth noting that the extreme concentration range of the same sample under different lysis conditions can reach approximately 14 times (approximately 3–44 ng / μL), highlighting the crucial role of optimizing lysis conditions in improving sperm DNA extraction efficiency.

[0028] Table 1

[0029]

[0030] 2. One-way ANOVA This embodiment evaluates the effects of SDS, PK, and DTT on the extraction of DNA from blue fox sperm. A one-way ANOVA was performed on each factor, and their F-values ​​were compared with... p The values ​​were compared, and the results are shown in Table 2.

[0031] The main effects of the three factors, SDS, PK, and DTT, were all statistically significant. p <0.005), where PK and DTT p The values ​​were all less than 0.001, indicating that their effects were extremely significant. Ranked by F-value, DTT had the strongest main effect (F=140.785), contributing the most to increasing the concentration of extracted DNA from blue fox sperm; PK was second (F=50.189); and SDS had a relatively weak effect (F=4.746). Therefore, in the lysis process of blue fox sperm samples, DTT concentration and dosage played the main roles, followed by PK, and lastly SDS.

[0032] Table 2

[0033] 3. Visualization and analysis of 3D scatter plots To further clarify the synergistic effects of SDS, PK, and DTT on the efficiency of blue fox sperm DNA extraction during sperm lysis, a three-dimensional scatter plot was used to visualize the relationship between SDS, DTT, PK at different concentrations and sperm DNA extraction concentration.

[0034] like Figure 1As shown, in the three-dimensional coordinate system, the X axis is the PK concentration (10-25 mg / mL), the Y axis is the DTT concentration (0.5-2 mol / L), and the Z axis is the SDS concentration (5%-20%), and the color of the point reflects the concentration of the sperm DNA extraction. It can be seen that the blue fox sperm DNA concentration gradually increases with the increase of the concentrations of PK, DTT and SDS, and reaches a peak in a certain concentration range; when the concentrations of the three lysis agents continue to increase, the extraction amount of some combinations decreases, indicating that too high concentration will inhibit the extraction of blue fox sperm DNA.

[0035] The optimal blue fox sperm sample lysis condition finally screened is 15% SDS + 20 mg / mL PK + 1.5 mol / L DTT; under the above optimal blue fox sperm sample lysis condition, the average value of the DNA extraction concentration of the samples numbered H1-H6 reaches 42.911 ± 0.939 ng / μL, corresponding to the most significant red high value point in the figure, indicating that the extraction efficiency of the blue fox sperm DNA is best under this condition.

[0036] It can be seen that although the three factors of SDS, PK and DTT play different roles in the lysis process of the blue fox sperm sample, the high-efficiency extraction method of the blue fox sperm DNA provided by the present application needs to realize the purpose of high-efficiency extraction of the blue fox sperm DNA through the synergistic effect of the three factors of SDS, PK and DTT.

[0037] 4. Agarose gel electrophoresis detection The samples numbered H1-H6 were subjected to DNA extraction under the optimal blue fox sperm sample lysis condition (15% SDS + 20 mg / mL PK + 1.5 mol / L DTT), and the obtained blue fox sperm DNA was subjected to 0.7% agarose gel electrophoresis detection, and the band image was recorded, and the specific operation steps were as follows: S1: Preparation of 0.7% agarose gel: weigh 0.35 g of agarose powder and add 50 mL of 1×TAE buffer; microwave heating until the agarose is completely dissolved, and a clear transparent solution appears; when the solution is cooled to about 60°C, add GelRed nucleic acid dye to a final concentration of 0.5 μg / mL, and mix thoroughly; pour the dyed agarose solution into the gel tray with the comb installed, and carefully remove the comb after solidification (about 20 min), and load into the electrophoresis tank; S2: Sample preparation and loading: take 5 μL from each DNA extraction product of the samples, add 1 μL of 6×Loading Buffer, and mix gently, taking care not to overflow or cross-contamination; at the same time, load the same volume of 10000 bp Marker DNA molecular weight standard; S3: electrophoresis running: add enough 1xTAE buffer to the electrophoresis tank to ensure that the gel is completely immersed; set the voltage to 80 V, and the electrophoresis time is about 50 min; S4: gel imaging and result recording: after the electrophoresis is completed, the gel is placed in the ultraviolet light transmission imaging system to take a picture, and the bands are observed.

[0038] As shown in the results Figure 2 , the DNA extraction samples numbered H1-H6 all have clear and continuous main bands in the high molecular weight region, the main band position is higher than 10000 bp, the band type is dense, and the dispersion is less, and no low molecular weight tailing or dispersion phenomenon is observed; it is shown that the length of the extracted DNA fragments is long, and no serious fragmentation or degradation occurs.

[0039] It can be seen that the efficient extraction method of blue fox sperm DNA provided by the application can not only significantly improve the concentration of blue fox sperm DNA, but also ensure the high structural integrity of the DNA product, fully meeting the subsequent high-throughput sequencing, specific amplification and other molecular biology research needs.

[0040] 5. Influence of blue fox sperm quantity on DNA concentration On the basis of this embodiment, the density of the blue fox semen sample numbered H1-H6 (obtained in Example 1) is adjusted, the sperm volume is fixed at 200 μL, and the influence of different initial blue fox sperm quantities on the DNA concentration of the blue fox semen is explored. The initial blue fox sperm density is set to be 5×10 6 / mL, 2.5×10 7 / mL, 5×10 7 / mL, 2.5×10 8 / mL and 5×10 8 / mL, respectively, and DNA extraction is carried out under the optimal blue fox sperm sample lysis conditions (15% SDS + 20 mg / mL PK + 1.5 mol / L DTT). The DNA concentration of each numbered sample under different sperm quantities is determined by using an ultramicro spectrophotometer, and the specific steps are the same as those in the above DNA concentration detection part.

[0041] As shown in Table 3, using the efficient extraction method of blue fox sperm DNA provided by the application, DNA extraction is carried out on the same blue fox sperm sample under different initial sperm quantities, and the obtained DNA concentrations are significantly different. When the number of blue fox sperm increases from 1×10 6 to 1×10 7 , the DNA concentration obtained by the samples H1-H6 gradually increases; and when the number of blue fox sperm is 1×10 7 , the DNA concentration obtained is the highest. However, when the number of sperm is further increased to 2×10 7 or even 1×108 The concentration of the obtained DNA is significantly reduced.

[0042] It can be seen that the high-efficiency extraction method of blue fox sperm DNA provided by the application is affected by the initial number of blue fox sperm. Too low input amount of blue fox sperm will result in insufficient DNA yield, and too high input amount of blue fox sperm will reduce the DNA yield due to excessive load of the lysis and extraction system. Therefore, it is calculated that when the total number of sperm in the blue fox semen is 1 x 10 7 The high-efficiency extraction method of blue fox sperm DNA provided by the application is best when the total number of sperm in the blue fox semen is 1 x 10

[0043] Table 3

[0044] 6. Effect of digestion processing time on DNA concentration On the basis of the present embodiment, the digestion processing time under the condition of 56°C was adjusted to explore the effect of digestion processing time on DNA concentration. The digestion processing time was set to 1 h, 2 h, 3 h and 4 h, respectively. DNA extraction was performed under the optimal lysis conditions of blue fox sperm samples (15% SDS + 20 mg / mL PK + 1.5 mol / L DTT), and the concentration of blue fox sperm DNA extracted from different numbered samples H1-H6 (obtained in Example 1) under different digestion times was determined using an ultramicro spectrophotometer. The specific steps are the same as the above DNA concentration detection part.

[0045] The results are shown in Table 4. Different digestion processing times have a significant effect on the concentration of the extracted blue fox sperm DNA. Specifically, when the digestion processing time is 1 h, the DNA concentration extracted from the blue fox semen samples numbered H1-H6 is relatively low, with an average value of 22.158 ± 2.598 ng / μL. This indicates that incomplete digestion processing leads to insufficient DNA release.

[0046] When the digestion processing time is extended to 2 h, the DNA concentration extracted from the blue fox semen samples numbered H1-H6 is significantly increased, reaching a maximum value of 42.920 ± 0.926 ng / μL. This indicates that when the digestion processing time is 2 h, the lysis and digestion effect on the blue fox semen sample is the most sufficient.

[0047] When the digestion processing time is extended to 3 h, the DNA concentration extracted from the blue fox semen samples numbered H1-H6 is slightly decreased, with an average value of 39.411 ± 1.376 ng / μL, but the overall level remains high.

[0048] When the digestion time is prolonged to 4 h, the DNA concentration extracted from the blue fox semen samples numbered H1-H6 is obviously decreased, and the average value is 30.815±4.144 ng / μL; it is shown that when the digestion time is prolonged to 3-4 h, DNA is degraded or the inhibiting factor is accumulated in the system, thereby affecting the DNA yield.

[0049] Therefore, the efficient blue fox sperm DNA extraction method provided by the application is best under the condition of digestion for 2 h at 56℃.

[0050] Table 4

[0051] Example 3: Phenol-chloroform-isoamyl alcohol extraction method for extracting blue fox sperm DNA 200 μL of the blue fox semen samples (sperm density is 5×10 7 / mL) numbered H1-H6 (obtained in Example 1) are mixed with an equal volume of 200 μL of TNE buffer (1×, pH 7.4) uniformly, 40 μL of SDS (concentration is 15% w / v), 20 μL of PK (concentration is 20 mg / mL) and 20 μL of DTT solution (concentration is 1.5 mol / L) with different concentrations are sequentially added, vortex oscillation is performed for 30 s, the components are fully mixed, and digestion is performed in a 56℃ constant temperature metal heater for 2 h, and the mixture is gently inverted and mixed once every 15 min during the digestion; the above mixed solution after digestion is subjected to DNA extraction by a phenol-chloroform-isoamyl alcohol extraction method.

[0052] The steps of the above phenol-chloroform-isoamyl alcohol extraction method for extracting blue fox sperm DNA are as follows: The above mixed solution after digestion is mixed with an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1, v / v / v) and is mixed uniformly by vigorous oscillation for 30 s; centrifugation is performed at 4℃ and 12000 rpm for 10 min, the supernatant is taken into a new centrifugal tube, and the above steps are repeated until the supernatant is transparent and no protein precipitate is generated; 2 times the volume of anhydrous ethanol and 1 / 10 times the volume of 3 mol / L sodium acetate (pH=5.2) are added to the supernatant and are gently inverted and mixed, precipitation is performed at-20℃ for 30 min, centrifugation is performed at 4℃ and 12000 rpm for 10 min, the supernatant is discarded, and the DNA precipitate is collected; the above precipitate is washed once with 70% ethanol, is centrifuged again, and the ethanol is discarded; the above precipitate is naturally air-dried at room temperature, and over-drying is avoided; finally, the DNA is dissolved by using a nuclease-free water solution, and the DNA is obtained.

[0053] Effect experiment: The concentration of the blue fox sperm DNA obtained from the different numbered samples was determined using an ultramicro spectrophotometer, and the specific steps were the same as those in the DNA concentration detection part of Example 2. The blue fox sperm DNA obtained from the different numbered samples was subjected to 0.7% agarose gel electrophoresis detection, and the band images were recorded, and the specific operation steps were the same as those in the agarose gel electrophoresis detection part of Example 2.

[0054] The results are shown in Figure 3 The DNA concentrations of the blue fox sperm samples H1-H6 extracted by the phenol-chloroform-isoamyl alcohol extraction method were 37.875 ng / μL, 38.243 ng / μL, 35.064 ng / μL, 39.987 ng / μL, 38.087 ng / μL, and 40.187 ng / μL, respectively, and the average DNA concentration was 38.241±1.849 ng / μL. Compared with the DNA concentration obtained by the silica gel column method (Example 2), the DNA concentration was slightly lower. The agarose gel electrophoresis detection graph showed that the DNA product band extracted by the phenol-chloroform-isoamyl alcohol extraction method was clear, uniform in migration, and had good integrity.

[0055] Example 4: Magnetic bead method for extracting blue fox sperm DNA 200 μL of blue fox semen samples (sperm density: 5×10 7 / mL) numbered H1-H6 (obtained in Example 1) were mixed with an equal volume of 200 μL TNE buffer (1×, pH 7.4) to obtain a homogeneous mixture. Different concentrations of 40 μL SDS (concentration: 15% w / v), 20 μL PK (concentration: 20 mg / mL), and 20 μL DTT solution (concentration: 1.5 mol / L) were added in sequence, and vortexed for 30 s to fully mix the components. The mixture was then placed in a 56°C constant temperature metal heater for digestion for 2 h, with gentle inversion every 15 min to mix.

[0056] The steps for extracting blue fox sperm DNA using the kit were as follows: The above-mentioned digestion-treated mixture and 350 μL of isopropanol were added to a centrifuge tube and mixed for 10 s. 15 μL of magnetic bead suspension GH was added and mixed for 1 min, and then allowed to stand for 9 min, with mixing every 3 min. To ensure that the magnetic beads were completely resuspended, they must be mixed before use. The centrifuge tube was placed on a magnetic stand for 30 s, and after the magnetic beads were completely adsorbed, the liquid was carefully removed. The mixture was centrifuged at 12000 rpm at 4°C for 10 min, and the supernatant was transferred to a new centrifuge tube to obtain the DNA.

[0057] Effect experiment: The DNA concentration of the blue fox sperm samples H1-H6 extracted above was determined using an ultramicro spectrophotometer, and the specific steps were the same as those in the DNA concentration detection part of Example 2. The DNA of the blue fox sperm samples H1-H6 extracted above was subjected to 0.7% agarose gel electrophoresis detection, and the band images were recorded, and the specific operation steps were the same as those in the agarose gel electrophoresis detection part of Example 2.

[0058] The results are shown in Figure 4 The DNA concentration of the blue fox sperm samples H1-H6 extracted by the magnetic bead method was 45.892 ng / μL, 42.952 ng / μL, 44.318 ng / μL, 44.162 ng / μL, 45.228 ng / μL, and 44.743 ng / μL, respectively, and the average DNA concentration was 44.549±1.006 ng / μL. The DNA concentration obtained by the magnetic bead method was slightly higher than that obtained by the silica gel column method (Example 2) and the phenol-chloroform-isoamyl alcohol extraction method (Example 3). The agarose gel electrophoresis detection chart showed that the DNA product band extracted by the magnetic bead method was clear, uniform in migration, and had good integrity.

[0059] Comparative Example 1 The blue fox semen samples (sperm density 5×10 7 / mL) numbered H1-H6 (obtained in Example 1) were subjected to DNA extraction using the Biaolebo sperm DNA extraction kit (purchased from Beijing Biaolebo Technology Co., Ltd.). The specific steps strictly followed the instructions, and the specific steps were as follows: 0.2 mL of the blue fox semen samples (sperm density 5×10 7 / mL) numbered H1-H6 (obtained in Example 1) were taken into a 1.5 mL centrifuge tube, 0.6 mL of sperm lysis solution was added, mixed by inverting 10 times, and incubated at 37°C for 10 min for complete lysis; 0.7 mL of the above mixture was added to a centrifugal adsorption column, placed in a collection tube, covered with a centrifugal adsorption column tube cap, and centrifuged at 14000 rpm at room temperature for 1 min, and the filtrate was discarded. The centrifugal adsorption column was placed back into the collection tube, covered with a centrifugal adsorption column tube cap, and centrifuged at 14000 rpm for 1 min. The centrifugal adsorption column was placed in a new 1.5 mL centrifuge tube, 100 μL of DNA elution solution was added to the center of the adsorption membrane, and the mixture was allowed to stand at room temperature for 1 min. The DNA solution was obtained by centrifugation at 14000 rpm for 30 s.

[0060] Effect experiment The concentration of the obtained blue fox sperm DNA of the different numbered samples was determined by using an ultramicro spectrophotometer, and the specific steps were the same as those in the DNA concentration detection part of Example 2.

[0061] The DNA concentrations of the blue fox sperm samples H1-H6 extracted by the Nuo Leibo sperm DNA extraction kit were 15.192 ng / μL, 16.986 ng / μL, 14.631 ng / μL, 24.109 ng / μL, 21.176 ng / μL, and 22.864 ng / μL, respectively, and the average DNA concentration was 19.160±4.081 ng / μL. It can be seen that the DNA concentrations of the blue fox sperm samples H1-H6 extracted by the Nuo Leibo sperm DNA extraction kit were significantly lower than the DNA concentrations obtained by the high-efficiency extraction method of the blue fox sperm DNA according to the present application, specifically including the silica gel column method (Example 2), the phenol-chloroform-isoamyl alcohol extraction method (Example 3), and the magnetic bead method (Example 4).

[0062] The above disclosed embodiments of the present application are only used to help explain the present application. The embodiments do not describe all the details, nor limit the present application to only the described embodiments. According to the content of the present application, many modifications and changes can be made. The present application selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present application, so that those skilled in the art can well understand and utilize the present application.

Claims

1. A method for efficient extraction of blue fox sperm DNA, characterized by, The method comprises the following steps: S1: mixing blue fox semen and TNE buffer according to a volume ratio of 1:1, adding into a DNA centrifuge tube to obtain a mixed solution; S2: sequentially adding SDS, PK and DTT solutions into the mixed solution obtained in S1, oscillating and mixing uniformly, and performing digestion treatment; S3: performing DNA extraction on the mixed solution after the digestion treatment in S2 to obtain blue fox sperm DNA.

2. The method of claim 1, wherein, The total number of sperm in the blue fox semen in S1 is 2 x 10 7 -2 x 10 6 .

3. The method of claim 2, wherein, The total number of sperm in the blue fox semen is 1 x 10 7 .

4. The method of claim 1, wherein, The concentration of the SDS in S2 is 15% (w / v), and the added amount is 40 μL.

5. The method of claim 1, wherein, The concentration of the PK in S2 is 20 mg / mL, and the added amount is 20 μL.

6. The method of claim 1, wherein, The concentration of the DTT in S2 is 1.5 mol / L, and the added amount is 20 μL.

7. The method of claim 1, wherein, The temperature of the digestion treatment in S2 is 56°C, and the reaction time is 2 h.

8. The method of claim 1, wherein, The method of the DNA extraction in S3 comprises but is not limited to a phenol-chloroform-isoamyl alcohol extraction method, a silica gel column method and a magnetic bead method.

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