Mouth wash DNA extraction method and application thereof in STR typing

Abstract

The invention relates to a mouthwash DNA extraction method and application thereof in STR typing. The method comprises the following steps: extracting genome DNA from mouthwash to perform STR locus amplification; no food is eaten after tooth brushing, and mouthwash is used for mouthwash at least one hour later; the gargling time is greater than or equal to 30s; when the mouth is rinsed, the mouth wash is fully in contact with the oral epithelium and gingival parts, a mouth wash sample is obtained and centrifuged through a centrifugal machine, and STR site amplification is achieved. And extracting a genome DNA solution by using a paramagnetic particle method. 10 ng of a genome DNA solution is taken as a PCR template, and chain polymerase amplification is carried out. The extracted sample can be stored at room temperature for 2 weeks, and downstream experiments can still be carried out. Meanwhile, under the condition of poor oral hygiene, genome DNA can be extracted for genotyping.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and DNA extraction technology, specifically relating to a low-cost method for extracting DNA from mouthwash and its application in STR typing. Background Technology

[0002] In general human understanding, children inherit many traits from their parents, such as appearance, skin color, and blood type; this is the phenomenon of heredity. The scientific basis for this phenomenon lies in the fact that all the genetic information of an organism is encoded in a biological macromolecule called deoxyribonucleic acid (DNA). DNA is the blueprint of life, and it exists in the vast majority of cells in the human body.

[0003] In biology, a person's complete DNA information (genome) is jointly inherited from both of their biological parents. Specifically, half of a child's genetic material comes precisely from their biological mother, and the other half precisely from their biological father. This semi-conservative inheritance of DNA is an objective and constant law, providing the most fundamental and reliable theoretical basis for scientifically identifying blood relationships between individuals, especially parent-child relationships. Therefore, the core of modern paternity testing technology is to make a judgment by comparing and analyzing the DNA characteristics between the child and the alleged father (or alleged mother).

[0004] The human genome contains approximately 3 billion base pairs, an enormous amount of information. Directly comparing the complete DNA sequences of two individuals is neither practical nor necessary. To achieve efficient and accurate individual identification and paternity determination, scientists have developed analytical techniques based on genetic markers. Among numerous genetic markers, short tandem repeats (STRs) technology has become the gold standard in global forensic science and paternity testing due to its high polymorphism, genetic stability, and ease of detection.

[0005] STRs are a class of DNA sequences widely found in the human genome. They are characterized by tandem repetitions of a core sequence consisting of 2 to 7 base pairs, such as "(GATA)(GATA)(GATA)...". The number of times the core sequence is repeated at the same STR locus (i.e., how many times "GATA" is repeated) usually varies among different individuals. This difference in the number of repetitions constitutes the "alleles" at that locus.

[0006] According to Mendel's laws of inheritance, an individual has two alleles at each STR locus, one from the mother and one from the father. Therefore, the standard procedure for paternity testing is as follows: Step 1: Simultaneously test multiple (usually 12 or more) STR loci of the individual being identified (children, suspected father).

[0007] Step 2: Compare one by one. At each STR locus, check whether one of the offspring's two alleles can be traced back to the mother (if the mother is involved in the identification) and the other can be traced back to the alleged father.

[0008] Step 3: Draw a conclusion. If all tested STR loci conform to genetic patterns, a parent-child relationship is supported; conversely, if multiple loci do not match, a parent-child relationship can be ruled out. This method, through the combined application of multiple loci, achieves extremely high accuracy.

[0009] Currently, the mainstream technology for STR analysis is "multiplex PCR amplification-capillary electrophoresis fragment analysis," which can detect and genotype multiple STR sites in a single, automated process.

[0010] To perform the above DNA analysis, a biological sample containing the DNA of the person being tested must first be obtained. In paternity testing practice, samples are generally divided into two main categories based on the ease of obtaining the sample and the quality of the DNA: Routine samples mainly include blood / bloodstains and oral swabs (oral mucosal cells). These samples are considered "ideal sources" for DNA extraction because they provide sufficient quantity and high quality DNA, and the collection process is relatively simple and standardized, resulting in a very high success rate for subsequent analysis.

[0011] Special samples: These refer to alternative specimens used in specific circumstances when regular samples are unavailable. For example, when the person being tested is unwilling to cooperate, is deceased, missing, or cannot be disturbed, the client may provide special specimens such as hair (with follicles), fingernails, toothbrushes, chewing gum, cigarette butts, tissues, or semen stains. These types of samples account for a significant proportion of paternity tests, providing a solution for many complex testing needs.

[0012] Although STR analysis technology itself is quite mature, its success is highly dependent on obtaining a "sufficient quantity and quality" of DNA from the sample. Special samples, due to their unique origins, face bottlenecks in DNA extraction and analysis that are difficult to overcome entirely with current technology. This directly leads to the risk of identification failure or inaccurate results. Specific problems include: Low DNA Quantity: The number of cells attached to samples such as toothbrushes, fingernails, and chewing gum is very limited, and the total amount of DNA that can be extracted is often in the picogram (pg) or even lower. When the amount of DNA template is too low, PCR amplification may fail completely or result in severe random effects (such as allele loss), leading to the inability to obtain effective STR typing results.

[0013] Severe DNA degradation: Special samples are often exposed to the environment for extended periods. Under the influence of factors such as bacteria, fungi, hydrolysis, and oxidation, long-chain DNA molecules break into many small fragments. STR analysis requires amplifying fragments within a specific length range (usually 100-500 bp). Severe degradation results in incomplete target fragments, making successful amplification and detection impossible.

[0014] PCR inhibitors are present: The sample carrier itself may contain substances that interfere with subsequent experiments. For example, tar in cigarette butts, chemicals in chewing gum, dyes on clothing, and humic acid in soil. If these substances are not effectively removed, they will inhibit the activity of key enzymes in the PCR reaction, leading to low amplification efficiency or even failure.

[0015] Exogenous DNA contamination: Special samples are highly susceptible to DNA contamination from other sources during formation, preservation, and transfer, such as items touched by multiple people or the growth of microorganisms. This results in extracted DNA containing DNA from other individuals, ultimately forming complex mixed STR patterns, which greatly complicates data interpretation and may even lead to erroneous conclusions.

[0016] Existing paternity testing methods generally include the following main steps: sample collection, DNA extraction, PCR amplification, product detection, and result analysis.

[0017] Phase 1: Sample Collection On-site sampling: Samples are collected by professionals from the testing center to ensure their authenticity and lack of contamination. The most commonly used sample types are oral swabs or blood. Oral swabs: A sterile cotton swab is used to swab the inside of the patient's mouth (inner cheek) to collect exfoliated oral mucosal cells. This method is non-invasive, fast, and safe, suitable for all ages, and is currently the mainstream choice. Blood: Usually, a few drops of blood are collected from the fingertip, or in special circumstances, a small amount of venous blood may be collected by a professional nurse.

[0018] Sample Marking and Recording: Collected samples are immediately placed in uniquely identified sample bags and sealed. Staff will photograph each sample for record-keeping and require the person being tested to provide fingerprint confirmation, ensuring that each sample corresponds to an individual's identity and preventing the risk of sample swapping.

[0019] The second stage: laboratory testing, which is the technical core of the entire identification process, typically includes the following steps: DNA Extraction: Researchers use specialized kits and methods to separate and purify DNA from collected samples (such as oral swabs or bloodstains), removing impurities such as proteins and lipids.

[0020] PCR amplification: Because the amount of extracted DNA is very small, polymerase chain reaction (PCR) technology is used to replicate a large number of specific DNA fragments. In paternity testing, the target of amplification is multiple short tandem repeat (STR) loci. These loci are highly polymorphic in the population and are key to individual identification and parentage determination.

[0021] Capillary electrophoresis and genotyping: Amplified PCR products are separated and detected using a capillary electrophoresis apparatus. The instrument precisely analyzes the allele type of each STR locus based on the different lengths of the DNA fragments. Ultimately, each individual will receive a DNA profile containing all the detected loci.

[0022] Phase Three: Data Analysis Genemapper 5 software was used to analyze capillary electrophoresis data to obtain the bp size of different STR loci amplified and to calculate the number of repeats at each STR locus to determine identity information. The resulting graphs showed whether different STR loci were completely amplified and the number of repeats at each STR locus. The different numbers of repeats could not only determine whether there was a kinship between individuals but also determine population information.

[0023] Question 1: Existing oral samples, such as saliva, while a routine testing method, cannot be preserved for long periods. Commercially available kits also have strict requirements regarding storage time; only fresh samples can yield sufficient genomic DNA. In samples stored for extended periods, the genomic DNA degrades significantly over time, making them unsuitable for large-scale population testing.

[0024] Question 2: Existing oral samples, such as saliva, require high oral hygiene from the test subjects. Highly contaminated saliva samples are prone to poor STR typing, such as missing peaks or spurious peaks.

[0025] Question 3: Existing oral samples, such as saliva, may exhibit incomplete amplification of STR sites if improperly preserved, such as through repeated freeze-thaw cycles.

[0026] Question 4: Commercially available magnetic bead kits for extracting genomic DNA are expensive. Summary of the Invention

[0027] This invention provides a method for extracting DNA from mouthwash and its application in STR typing, enabling the extracted samples to be stored at room temperature for two weeks and still suitable for downstream experiments. Furthermore, it allows for the extraction of genomic DNA for genotyping even under conditions of poor oral hygiene.

[0028] A method for extracting DNA from mouthwash includes the following steps: Step S1: Extract genomic DNA from mouthwash for STR site amplification; Step S2: Extract genomic DNA solution using magnetic beads.

[0029] Preferably, the specific process of step S1 of the present invention is as follows: after brushing teeth, do not eat, and rinse mouth with mouthwash at least 1 hour later; the rinsing time is greater than or equal to 30 seconds; when rinsing, allow the mouthwash to fully contact the oral epithelium and gums, obtain a mouthwash sample, and centrifuge it to achieve STR site amplification.

[0030] Preferably, in this invention, 10 ml of mouthwash sample is added to a 50 ml sterile centrifuge tube, centrifuged at 4000 rpm for 10 min at low temperature, and the bottom of the centrifuge tube is observed to see if there is a buildup of misty cells; if there is no obvious cell buildup at the bottom, the mouthwash sample needs to be prepared again.

[0031] Preferably, the specific process of step S2 of the present invention is as follows: Step S21: After pouring out all the supernatant from the centrifugation in step S1, add 400 µl of PBS solution; gently pipette the cells to mix them with the PBS solution. Step S22: Add 10 µl of 10% SDS solution to the mixed cell suspension and incubate in a water bath or metal bath at 37°C to perform preliminary cell membrane lysis. Step S23: Add 400 µl of lysis buffer and 20 µl of 10 mg / ml proteinase K solution, vortex for 15 seconds to ensure uniform mixing; place in a 65°C water bath for 25-30 minutes until there are no obvious tissue clumps in the solution; Step S24: Add 10 µl of magnetic beads and 400 µl of binding solution to a centrifuge tube, shake well for 15 seconds, place on a magnetic rack and let stand for 2 minutes until the magnetic beads are evenly adsorbed on the magnetic rack, then discard the supernatant. Step S25: Add 800 µl of 80% ethanol, shake in a mixer for 15 seconds, place the centrifuge tube on a magnetic rack for 2 minutes, and discard the supernatant after the magnetic beads have been completely adsorbed onto the magnetic rack. Step S26: Repeat step S25; after removing the supernatant from the centrifuge tube, let it air dry at room temperature for 15-25 minutes to allow all the ethanol to evaporate. Alternatively, place it in a 55°C oven for 5 minutes to allow all the ethanol to evaporate.

[0032] Step S27: Add 30 µl of TE solution, ensuring that the magnetic beads are completely coated with the TE solution, and incubate in a water bath or metal bath at 65°C for 5 minutes.

[0033] Step S28: Place the centrifuge tube on a magnetic rack and wait for the magnetic beads to adhere to the rack. The resulting supernatant is the solution containing genomic DNA. It can be stored at -20°C for long-term storage.

[0034] Preferably, if RNA is not required, RNase is added in step S23 and incubated at 37°C for 15 minutes.

[0035] Preferably, the lysis buffer of the present invention comprises Tris-HCl, EDTA, Tween-20, DTT, and deionized water, and the pH value after preparation is 8.0.

[0036] Preferably, the binding solution of the present invention includes Guanidine hydrochloride, Isopropanol, NaCl, Tris-HCl, and DMSO.

[0037] Preferably, the eluent of the present invention is 80% ethanol.

[0038] An application of genomic DNA obtained by mouthwash DNA extraction in STR typing: 10 ng of genomic DNA was used as a PCR template for chain polymerase amplification.

[0039] Preferably, the amplified locus of the present invention is at least one of D3S1358, vWA, D7S820, CSF1PO, PentaE, D8S1179, D21S11, D16S539, D2S1338, PentaD, D19S433, TH01, D13S17, TPOX, D18S51, D6S1043, AMEL, D1S1656, D5S818, D12S391, and FGA.

[0040] The technical solution of this invention has the following advantages compared with the prior art: 1. Invasive sampling is unpleasant. The sampling method of this invention is simple, quick and does not cause aversion.

[0041] 2. Existing sampling methods have short storage times and severe degradation, resulting in low efficiency of STR amplification in downstream experiments and peak loss in CE detection. In contrast, samples extracted using this method can be stored at room temperature for two weeks, and the obtained STR profiles are complete with good peak values, allowing for continued downstream experiments.

[0042] 3. Existing oral samples, such as saliva, may exhibit incomplete STR amplification if improperly stored, such as after repeated freeze-thaw cycles. However, this method can still extract trace amounts of DNA for genotyping from repeatedly frozen-thawed samples, amplifying complete STR maps with good peak shapes.

[0043] 4. Even in cases of poor oral hygiene, genomic DNA can still be extracted for genotyping, and complete STR images with good peak shapes can still be amplified. Detailed Implementation

[0044] The technical solution of the present invention will be described in detail below: A method for extracting DNA from mouthwash includes the following steps: Step S1: Extract genomic DNA from mouthwash for STR site amplification: After brushing your teeth, do not eat; ideally, wait one hour. Use sterile or purified water for rinsing. Rinse for at least 30 seconds, otherwise, the number of oral epithelial cells will be insufficient. Ensure the mouthwash fully contacts the oral epithelium and gums. Add 10 ml of the mouthwash sample to a 50 ml sterile centrifuge tube and centrifuge at 4000 rpm for 10 minutes. Observe the bottom of the centrifuge tube for any mist-like cell accumulation. If there is insufficient cell accumulation, increase the centrifuge speed or extend the centrifugation time. If there is no obvious cell accumulation at the bottom, prepare fresh mouthwash to avoid insufficient genomic DNA, which could lead to the failure of downstream experiments.

[0045] Step S2: Extract genomic DNA solution using magnetic beads: Step S21: After discarding all the supernatant from centrifugation, add 400 µl of PBS solution. Gently pipette the cells to mix them with the PBS solution.

[0046] Step S22: Add 10 µl of 10% SDS solution to the mixed cell suspension and incubate in a water bath or metal bath at 37°C to perform preliminary cell membrane lysis.

[0047] Step S23: Add 400 µl of lysis buffer and 20 µl of 10 mg / ml proteinase K solution. Vortex for 15 seconds to ensure homogeneity. Incubate in a 65°C water bath for 25-30 minutes, or until no obvious tissue clumps remain in the solution.

[0048] If the downstream experiment does not require RNA, add RNase in this step and incubate at 37°C for 15 minutes.

[0049] Step S24: Add 10µl of magnetic beads and 400µl of binding solution to a centrifuge tube, shake well for 15 seconds, place on a magnetic rack and let stand for 2 minutes until the magnetic beads are evenly adsorbed on the magnetic rack, then discard the supernatant.

[0050] Step S25: Add 800µl of 80% ethanol, shake in a mixer for 15 seconds, place the centrifuge tube on a magnetic rack for 2 minutes, and discard the supernatant after the magnetic beads have been completely adsorbed onto the magnetic rack.

[0051] Step S26: Repeat the previous step; after removing the supernatant from the centrifuge tube, let it air dry at room temperature for 15-25 minutes to allow all the ethanol to evaporate. Alternatively, place it in a 55°C oven for 5 minutes to allow all the ethanol to evaporate.

[0052] Step S27: Add 30µl of TE solution, ensuring that the magnetic beads are completely coated with the TE solution, and incubate in a water bath or metal bath at 65°C for 5 minutes.

[0053] Step S28: Place the centrifuge tube on a magnetic rack and wait for the magnetic beads to adhere to the rack. The resulting supernatant is the solution containing genomic DNA. It can be stored at -20°C for long-term storage.

[0054] The lysis buffer formulation of this invention includes: Tris-HCl, EDTA, Tween-20, DTT, and deionized water. After preparation, the pH value is adjusted to 8.0.

[0055] The binding solution formulation of this invention includes: Guanidine hydrochloride, Isopropanol, NaCl, Tris-HCl, and DMSO.

[0056] The eluent of this invention is 80% ethanol.

[0057] The magnetic beads of this invention are: Shanghai Sangon Silicon Hydroxyl Magnetic Beads.

[0058] An application of genomic DNA obtained using a mouthwash DNA extraction method in STR typing was described. 10 ng of genomic DNA was used as a template for PCR (which utilizes the property of DNA double-stranded DNA to dissociate at high temperatures and the ability of DNA polymerase to synthesize complementary strands along primer directions based on a template, enabling exponential amplification of specific DNA fragments in vitro). Chain polymerase amplification was performed. Amplified loci (D3S1358, vWA, D7S820, CSF1PO, PentaE, D8S1179, D21S11, D16S539, D2S1338, PentaD, D19S433, TH01, D13S17, TPOX, D18S51, D6S1043, AMEL, D1S1656, D5S818, D12S391, FGA) were analyzed. After amplification, the sample was sent for capillary electrophoresis to obtain a genotype using Genemapper 5.

[0059] From chain polymerase amplification to capillary electrophoresis detection and Genemapper 5 genotyping, all data in this experiment were sent to a third-party biotechnology company.

[0060] After extracting mouthwash samples from volunteer 1's oral cavity, genomic DNA extracted using the Shanghai Sangon Magnetic Bead Micro-Genomic DNA Extraction Kit was subjected to STR typing. Table 1 shows the amplification results of the genomic DNA extracted using the Shanghai Sangon Magnetic Bead Micro-Genomic DNA Extraction Kit at each amplification locus.

[0061] Table 1

[0062] As can be seen from Table 1, the classification is good, the size of each peak is normal, and there are no extraneous peaks.

[0063] After extracting mouthwash samples from the oral cavity of volunteer 2, genomic DNA extracted using this method was used for STR typing. Table 2 shows the amplification results of genomic DNA obtained using the DNA extraction method of this invention at each amplification locus.

[0064] Table 2

[0065] As can be seen from Table 2, the typing is good, the peak sizes are normal, there are no impurities, and the quality is comparable to commercially available reagent kits.

[0066] Volunteer 3 did not brush his teeth after waking up and ate breakfast to simulate an unclean oral environment. Contaminated mouthwash samples were extracted from Volunteer 3's mouth. The genomic DNA of the contaminated samples was STR-generated using this method. Table 3 shows the amplification results of the genomic DNA obtained by the DNA extraction method of this invention at each amplification locus.

[0067] Table 3

[0068] As can be seen from Table 3, the typing is good, the peak sizes are normal, there are no impurities, and the quality is comparable to commercially available reagent kits.

[0069] Volunteer 3 did not brush their teeth after waking up and ate breakfast to simulate an unclean oral environment. Contaminated saliva samples were extracted from Volunteer 3's mouths, and STR genomic typing of the contaminated samples was performed using the Ezup column-based saliva genomic DNA extraction kit. Table 4 shows the amplification results of the genomic DNA extracted using the Ezup column-based saliva genomic DNA extraction kit at each amplification locus.

[0070] Table 4

[0071] As shown in Tables 3 and 4, this method can achieve complete amplification patterns, good peak shapes, and no extraneous peaks for samples with poor oral environments and contamination. However, saliva samples and commercially available kits may exhibit peak loss and spurious peaks, which are not necessarily due to weak peak signals.

[0072] To ensure oral hygiene for volunteer 4, a mouthwash sample was collected from volunteer 4's mouth 2 hours after brushing. The sample was left at room temperature for 14 days, and the STR typing of the mouthwash after 14 days was performed using this method. Table 5 shows the amplification results of genomic DNA obtained by the DNA extraction method of this invention at each amplification locus.

[0073] Table 5

[0074] To ensure oral hygiene for volunteer 4, saliva samples were collected from volunteer 4's mouth 2 hours after brushing. After the samples were left at room temperature for 14 days, STR typing of the extracted genomic DNA was performed using the Ezup column-type saliva genomic DNA extraction kit. Table 6 shows the amplification results of the genomic DNA extracted using the Ezup column-type saliva genomic DNA extraction kit at each amplification locus.

[0075] Table 6

[0076] As shown in Tables 5 and 6, this method can achieve complete amplification patterns, good peak shapes, and no extraneous peaks for samples that have been stored for a long time. However, saliva samples and commercially available kits cannot perform STR genome amplification experiments on samples that have been stored for a long time.

[0077] To ensure oral hygiene for volunteer 5, a mouthwash sample was collected from volunteer 5's mouth 2 hours after brushing. This sample was then subjected to three freeze-thaw cycles to simulate improper storage conditions. Genomic DNA was extracted from the repeatedly freeze-thawed mouthwash sample using this method, and STR typing was performed. Table 7 shows the amplification results of the genomic DNA obtained using the DNA extraction method of this invention at each amplification locus.

[0078] Table 7

[0079] To ensure oral hygiene for volunteer 5, saliva samples were collected from volunteer 5's mouth 2 hours after brushing. These samples were then subjected to three freeze-thaw cycles to simulate improper storage conditions. Genomic DNA was extracted from the repeatedly frozen-thawed saliva samples using the Ezup column-based saliva genomic DNA extraction kit, and STR typing was performed. Table 8 shows the amplification results of the genomic DNA extracted using the Ezup column-based saliva genomic DNA extraction kit at each amplification locus.

[0080] Table 8

[0081] As shown in Tables 7 and 8, for samples that have been repeatedly frozen and thawed under poor storage conditions, this method can achieve complete amplification patterns, good peak shapes, and no extraneous peaks. However, saliva samples and commercially available kits may exhibit partial genomic failures, numerous non-specific amplification peaks, and generally weak signals in the amplified STR genome.

[0082] To ensure oral hygiene for volunteer 7, saliva samples were collected from their mouths two hours after brushing. Genomic DNA was extracted using the Dzup Genomic DNA Rapid Extraction Kit for STR typing. Animal tissue was lysed under high pH conditions using guanidine salt plasma solvent and denaturing agents, and RNA was partially hydrolyzed. Genomic DNA was then rapidly obtained through steps including ethanol precipitation. Although complete STR locus maps could be amplified, the process was time-consuming, involved multiple high-speed centrifuges, and contained highly corrosive compounds in the Dzup reagents, resulting in a high risk factor during operation.

[0083] The DNA extraction method of this invention yields a higher concentration of genomic DNA compared to column extraction. It is also simple to operate, economical, and free of harmful organic reagents. When the sample has a low genomic content, the magnetic bead method of this invention can extract DNA more effectively. Furthermore, the magnetic bead method of this invention is much cheaper than commercially available genomic DNA extraction kits.

Claims

1. A method for extracting DNA from mouthwash, characterized in that... Includes the following steps: Step S1: Extract genomic DNA from mouthwash for STR site amplification; Step S2: Extract genomic DNA solution using magnetic beads.

2. The method for extracting DNA from mouthwash according to claim 1, characterized in that... The specific process of step S1 above is as follows: Do not eat after brushing your teeth, and rinse your mouth with mouthwash at least 1 hour later; the rinsing time is greater than or equal to 30 seconds; when rinsing, make sure that the mouthwash comes into full contact with the oral epithelium and gums, obtain a mouthwash sample, and centrifuge it to achieve STR site amplification.

3. The method for extracting DNA from mouthwash according to claim 2, characterized in that... Add 10 ml of mouthwash sample to a 50 ml sterile centrifuge tube, centrifuge at 4000 rpm for 10 min, and observe the bottom of the centrifuge tube for any accumulation of mist-like cells. If there is no obvious cell accumulation at the bottom, a new mouthwash sample needs to be prepared.

4. The method for extracting DNA from mouthwash according to claim 2, characterized in that... The specific process of step S2 above is as follows: Step S21: After pouring out all the supernatant from the centrifugation in step S1, add 400 µl of PBS solution; gently pipette the cells to mix them with the PBS solution. Step S22: Add 10 µl of 10% SDS solution to the mixed cell suspension and incubate in a water bath or metal bath at 37°C to perform preliminary cell membrane lysis. Step S23: Add 400 µl of lysis buffer and 20 µl of 10 mg / ml proteinase K solution, vortex for 15 seconds to ensure uniform mixing; place in a 65°C water bath for 25-30 minutes until there are no obvious tissue clumps in the solution; Step S24: Add 10 µl of magnetic beads and 400 µl of binding solution to a centrifuge tube, shake well for 15 seconds, place on a magnetic rack and let stand for 2 minutes until the magnetic beads are evenly adsorbed on the magnetic rack, then discard the supernatant. Step S25: Add 800µl of 80% ethanol, shake in a mixer for 15 seconds, place the centrifuge tube on a magnetic rack for 2 minutes, and discard the supernatant after the magnetic beads have been completely adsorbed onto the magnetic rack. Step S26: Repeat step S25; after removing the supernatant from the centrifuge tube, let it air dry at room temperature for 15-25 minutes to allow all the ethanol to evaporate; or place it in an oven at 55°C for 5 minutes to allow all the ethanol to evaporate. Step S27: Add 30 µl of TE solution to ensure that the magnetic beads are completely coated with TE solution, and incubate in a water bath or metal bath at 65°C for 5 minutes. Step S28: Place the centrifuge tube on the magnetic rack and wait for the magnetic beads to adhere to the rack; the resulting supernatant is a solution containing genomic DNA; it can be stored at -20°C for a long time.

5. The method for extracting DNA from mouthwash according to claim 4, characterized in that... If RNA is not required, add RNase in step S23 and incubate at 37°C for 15 minutes.

6. The method for extracting DNA from mouthwash according to claim 4, characterized in that... The lysis buffer consists of Tris-HCl, EDTA, Tween-20, DTT, and deionized water, with a final pH of 8.

0.

7. The method for extracting DNA from mouthwash according to claim 2, characterized in that... The binding solutions include Guanidine hydrochloride, Isopropanol, NaCl, Tris-HCl, and DMSO.

8. The method for extracting DNA from mouthwash according to claim 2, characterized in that... The eluent was 80% ethanol.

9. An application of genomic DNA obtained using the mouthwash DNA extraction method of claim 1 in STR typing, characterized in that... Use 10 ng of genomic DNA solution as a PCR template for chain polymerase amplification.

10. The application of genomic DNA in STR typing according to claim 9, characterized in that... The amplified locus is at least one of D3S1358, vWA, D7S820, CSF1PO, PentaE, D8S1179, D21S11, D16S539, D2S1338, PentaD, D19S433, TH01, D13S17, TPOX, D18S51, D6S1043, AMEL, D1S1656, D5S818, D12S391, and FGA.

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