A method for extracting DNA from trace skin samples

Abstract

This invention discloses a method for extracting DNA from trace skin samples, belonging to the field of biotechnology. The method utilizes a lysis buffer to efficiently lyse keratinocytes, employs a specific binding buffer to remove PCR inhibitors such as sebum and cosmetics, and combines an optimized silica membrane adsorption column with a multi-step washing process to achieve efficient extraction and purification of DNA from trace amounts of highly degraded facial skin samples containing complex inhibitors. This method offers high DNA yield, high purity, and is simple and rapid to operate, making it particularly suitable for downstream molecular biological analysis of trace DNA samples in fields such as medicine, personalized medicine, and dermatology.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a non-invasive, highly sensitive DNA extraction method. Specifically, it is a method for the efficient extraction and purification of DNA from trace amounts of epithelial cells shed from the surface of facial skin. Background Technology

[0002] With the rapid development of medicine, personalized medicine, skin microbiome, and cosmetic skincare, the demand for non-invasive DNA sampling and testing is increasing. Facial skin is the main part of the human body that comes into direct contact with the external environment. Its surface microbial community (skin microbiome), sebum secretion, and metabolic products are important indicators reflecting an individual's health status, skin condition, and even certain diseases. Precise analysis of facial skin samples is of great value in fields such as cosmetic efficacy evaluation, dermatological research, microbial community analysis, personalized health management, and telomere research.

[0003] Currently, common methods for facial skin sampling include swabbing, tape peeling, and skin scraping. However, for extremely small (nanogram-level) and potentially highly degraded DNA samples from facial skin, traditional extraction methods (such as phenol-chlorine extraction and conventional column membrane extraction procedures) have significant shortcomings: 1. Extremely small sample volume: Facial swabbing yields a limited number of cells, resulting in low total DNA and low recovery rates with conventional methods; 2. Numerous inhibitors: Samples often contain PCR inhibitors from skincare products, cosmetics, sebum, and environmental pollutants, severely impacting downstream analysis; 3. DNA degradation: Environmental exposure may lead to a certain degree of DNA degradation; 4. Cumbersome operation: Traditional methods involve numerous steps and are time-consuming, easily resulting in the loss of trace samples during transfer. Therefore, there is an urgent need to research and develop a method that can efficiently, rapidly, and specifically extract high-quality DNA from complex, trace facial skin samples. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, the present invention aims to provide a highly efficient, stable, and interference-resistant method for extracting DNA from trace samples of facial skin. This method optimizes each step of lysis, binding, washing, and elution, significantly improving the yield and purity of DNA. It is particularly suitable for downstream molecular biology applications such as high-throughput sequencing (NGS), PCR, and STR analysis.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] This invention provides a method for extracting DNA from trace skin samples, comprising the following steps: (1) Collect exfoliated epithelial cells from the skin and transfer them to a preservation solution, then mix thoroughly: (2) Add lysis buffer to the sample solution obtained in step (1) and grind it. Add proteinase K, mix well, and incubate at 50-65°C for 4-16 hours with shaking. Add binding buffer and anhydrous ethanol, mix well, and let stand at room temperature for 3-30 minutes to ensure complete cell lysis and complete DNA release. (3) Transfer the lysed sample solution obtained in step (2) to a silica membrane adsorption column, place the adsorption column in a collection tube, centrifuge, discard the collection tube and the filtrate; (4) Place the adsorption column in a new collection tube and add a washing buffer containing a high concentration of guanidine salt and anhydrous ethanol to the adsorption column. Centrifuge, discard the collection tube and the filtered waste liquid. Place the adsorption column in a new collection tube and add an elution buffer containing anhydrous ethanol and a low concentration of buffer salt to the adsorption column. Centrifuge, discard the collection tube and the filtered waste liquid. (5) Place the adsorption column obtained in step (4) into a new collection tube, centrifuge for 2-5 minutes to completely dry the membrane of the adsorption column, place the adsorption column into a new collection tube, add sterile ultrapure water or distilled water to the adsorption column, incubate at room temperature for 3-10 minutes, centrifuge for 30-180 seconds, collect the filtrate, and you will get the product.

[0007] Based on the above technical solution, further, the specific process of step (1) is as follows: use a microfiber swab or sampling tape pre-moistened with physiological saline to gently and evenly wipe a specific area of ​​the subject's face (including the cheeks and forehead) to collect exfoliated epithelial cells, and immediately put the obtained swab head or tape into the preservation solution, or directly transfer the liquid sample into the preservation solution and mix evenly.

[0008] Based on the above technical solution, the composition of the preservation solution in step (1) is: 5~15 mM Tris-HCl, 0.5~1.5 mM EDTA, the remainder is water, and the pH is 7.5~8.5.

[0009] Based on the above technical solution, the composition of the lysis solution in step (2) is: 0.5~1.5% SDS, 10~30 mM EDTA, 5~15 mM Tris-HCl, with the remainder being water, and pH 7.5~8.5.

[0010] Based on the above technical solution, further, the composition of the binding buffer in step (2) is: 3~5M guanidine isothiocyanate, 20~40% isopropanol, 0.05~0.5M Tris-HCl, with the remainder being water, pH 6.0~7.0.

[0011] The binding buffer of this adsorption column is specially formulated to maximize the adsorption of trace amounts of DNA onto the silica membrane under high-concentration salt (guanidine isothiocyanate) and specific pH conditions, while allowing residual inhibitors to pass through.

[0012] Based on the above technical solution, further, the volume ratio of the sample solution, lysis buffer, binding buffer and anhydrous ethanol in step (2) is 5:1:1:1~1:1:1:1.

[0013] Based on the above technical solution, further, the final concentration of proteinase K in step (2) is 0.05~0.5 mg / mL.

[0014] Based on the above technical solution, further, the rotational speed of the oscillation in step (2) is 500~1500 rpm.

[0015] Based on the above technical solution, further, the silica membrane adsorption column in step (3) is a QIAamp MinElute column; the centrifugation conditions are: centrifugation at 7500~8500 rpm for 30~180 seconds.

[0016] Based on the above technical solution, further, the washing buffer in step (4) is composed of: guanidine hydrochloride 4~5M, Tris-HCl 20~40mM, NaCl 80~120mM, anhydrous ethanol 50~60%, and the remainder is water.

[0017] Based on the above technical solution, further, the composition of the elution buffer solution in step (4) is: Tris-HCl 5~15mM, EDTA 0.1~1mM, anhydrous ethanol 60~70%, and the remainder is water.

[0018] Based on the above technical solution, further, the centrifugation conditions in step (4) are: centrifugation at 13000~15000 rpm for 30~180 seconds.

[0019] Based on the above technical solution, further, the centrifugation speed in step (5) is 13000~15000 rpm.

[0020] Based on the above technical solution, further, after step (5) is completed, the obtained filtrate is added back to the adsorption column for a second centrifugation and elution, and the filtrate is collected to obtain the final product.

[0021] The beneficial effects of this invention are as follows: 1. High sensitivity and high yield: Through an optimized lysis and binding system, DNA can be effectively recovered from trace samples as small as one swab.

[0022] 2. High Purity: A unique inhibitor removal process and multi-step washing regimen effectively remove complex inhibitors such as sebum and cosmetic residues, resulting in DNA with high OD... 260 / 280 The ratio is stable between 1.7 and 2.1, and can be directly used in sensitive downstream applications.

[0023] 3. Wide range of applications: The extracted DNA is suitable for various precision analyses such as telomere analysis, SNP genotyping, qPCR quantification, and metagenomic sequencing. Attached Figure Description

[0024] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0025] Figure 1 : A schematic diagram of the DNA extraction method described in this invention.

[0026] Figure 2 The DNA integrity detection map obtained in Example 1 of this invention.

[0027] Figure 3 Electrophoresis diagram of DNA extracted in Example 1 of this invention subjected to PCR amplification (the amplified fragment is the human β-actin gene).

[0028] Figure 4 The DNA integrity detection map obtained from experimental group sample 1 in Example 2 of this invention.

[0029] Figure 5 The DNA integrity detection map obtained from experimental group sample 2 in Example 2 of this invention.

[0030] Figure 6 The DNA integrity detection map obtained from experimental group sample 3 in Example 2 of this invention.

[0031] Figure 7 The DNA integrity detection map obtained from experimental group sample 4 in Example 2 of this invention. Detailed Implementation

[0032] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0033] Example 1: This embodiment provides a method for DNA extraction based on a trace sample of facial skin, including the following steps: 1. Preparation and Requirements for Subjects Before Sampling: To ensure the quality and purity of the sample DNA and avoid exogenous contamination and interference from inhibitors, the following requirements and pretreatment procedures must be implemented for the subjects before sample collection: (1) Cleaning requirements: Subjects must not use any skin care products, cosmetics, sunscreen or foundation or other facial coverings for at least 6 hours before sampling; (2) Drug contraindications: Within one week prior to sampling, no topical antibiotics, antifungal drugs, retinoids or other prescription or over-the-counter drugs that may significantly alter the skin microenvironment may be used on the face (unless it is the study subject itself).

[0034] (3) Health status: Subjects must declare that they have no active facial skin diseases (such as acne breakouts, eczema, dermatitis, etc.), no systemic infections, and have not taken systemic antibiotics (within the past month).

[0035] (4) Facial pretreatment: Before formal sampling, the operator should use sterile gauze or medical cotton pads moistened with sterile ultrapure water to gently clean the facial area of ​​the subject to be sampled (such as cheeks or forehead) to remove some environmental adhering substances and excess sebum, and let it air dry or use sterile airflow to dry it; this step can significantly reduce the concentration of inhibitors in the sample.

[0036] 2. Sample collection and transfer: (1) Use a saline-moistened microfiber swab to gently and evenly wipe specific areas of the subject’s face (such as cheeks, forehead, etc.) to collect exfoliated epithelial cells.

[0037] (2) Immediately cut the collected swab head into a 1.5 ml microcentrifuge tube containing a special preservation solution (components: 10 mM Tris-HCl, 1 mM EDTA, the remainder is water, pH 8.0), and vortex to mix.

[0038] 3. DNA extraction procedure: (1) Add 200µl of lysis buffer (components: 1% SDS, 20mM EDTA, 10mM Tris-HCl, balance water, pH 8.0) to the above-mentioned swab-containing preservation solution (400µl), and then grind it with the matching grinding pestle for about 30 seconds; (2) Add 20 µl of proteinase K (final concentration approximately 0.1 mg / ml), vortex for 15 seconds, and then centrifuge the tube briefly (>5000 rpm, 5 seconds) to collect droplets from the tube wall. Then place the 1.5 ml centrifuge tube in a 56°C constant temperature shaker and incubate at 1200 rpm for 4-16 h until the sample is completely lysed; the specific time can be determined according to the sample quality.

[0039] (3) After incubation, centrifuge the centrifuge tube briefly, and add 200 μl of binding buffer (components: 4M guanidine isothiocyanate, 30% isopropanol, 0.1M Tris-HCl, the remainder is water, pH 6.4) and 200 μl of anhydrous ethanol to the lysis product in sequence. Vortex mix for 15 seconds immediately, and let stand at room temperature for 5 minutes to allow the cells to lyse more thoroughly.

[0040] (4) Briefly centrifuge the centrifuge tube to remove the droplets inside the cap, and carefully transfer all the mixture from step (3) to the silica membrane adsorption column (QIAamp MinElute column) used in this method. Place the adsorption column in a 2ml collection tube, centrifuge at 8000rpm for 1 minute, and discard the collection tube and the waste liquid therein.

[0041] (5) Place the adsorption column in a new 2ml collection tube and add 500µl of washing buffer containing high concentration of guanidine salt and anhydrous ethanol (guanidine hydrochloride 4.5M, Tris-HCl 30mM, NaCl 100mM and 55% anhydrous ethanol). Centrifuge at 14000rpm for 1 minute, discard the collection tube and its waste liquid, and remove impurities such as proteins, salts, residual lysis buffer and some organic solvents.

[0042] (6) Place the adsorption column in a new 2ml collection tube and add 500µl of elution buffer solution containing ethanol and low concentration buffer salt (Tris-HCl 10mM, EDTA 0.5mM and 65% anhydrous ethanol) to the adsorption column. Centrifuge at 14000rpm for 1 minute and discard the collection tube and the filtered waste liquid.

[0043] (7) Place the adsorption column in a new 2ml collection tube and centrifuge at 14000rpm for 3 minutes to allow the membrane to dry completely.

[0044] (8) Place the adsorption column into a new 1.5 ml labeled collection tube, add 35 µl of distilled water, seal the cap and incubate at room temperature (15-25 °C) for 5 minutes.

[0045] (9) Centrifuge the centrifuge tube with the adsorption column at 14000 rpm for 1 minute, and the collected liquid is the extracted DNA.

[0046] 4. DNA quality testing (1) Concentration and purity detection: Take 2 μl of DNA elution buffer and detect it using a NanoDrop2000 micro-volume UV spectrophotometer (Thermo Fisher Scientific). The instrument directly measures the absorbance (OD value) of the sample at 260 nm and 280 nm, and automatically calculates the DNA concentration (ng / μl) and OD260 according to the Lambert-Beer law. nm / 280nm Ratio (purity index).

[0047] (2) Integrity test: Dilute the above DNA elution buffer to a concentration of 1 ng / µl with 1×Dilution buffer, and take 10 µl for analysis in the Qsep100 fully automated nucleic acid and protein analysis system. The results are as follows: Figure 2 As shown in the figure, the results indicate that the sample fragments have high integrity.

[0048] (3) Specificity detection: 2 μl of the extracted DNA elution buffer was used for PCR amplification of the human internal reference gene β-actin, and the amplification product was analyzed by gel electrophoresis. The results are as follows: Figure 3 As shown, according to Figure 3 The gel electrophoresis image shows that the bands are clear and bright, with no interference from inhibitors.

[0049] Example 2: Comparison of Extraction Results Twenty facial swab samples from different healthy volunteers were processed using the method described in Example 1 (experimental group) and a commercially available DNA extraction kit (Quick-DNA™ MiniPrep Plus Kit, control group). Two swabs were collected from each volunteer. Sample collection followed the pretreatment requirements of Example 1. After extraction, samples were analyzed using a NanoDrop2000 micro-volume UV spectrophotometer. The results showed that the DNA extraction concentration in the experimental group was higher than that in the control group. This indicates that the method of the present invention has significant advantages in extracting trace amounts of DNA from the face.

[0050] Table 1. Comparison of DNA extraction concentration between the experimental group and the control group.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for extracting DNA from trace skin samples, characterized in that, Includes the following steps: (1) Collect exfoliated epithelial cells from the skin and transfer them to a preservation solution, then mix thoroughly: (2) Add lysis buffer to the sample solution obtained in step (1) and grind it. Add proteinase K, mix well, and incubate at 50-65°C for 4-16 hours with shaking. Add binding buffer and anhydrous ethanol, mix well, and let stand at room temperature for 3-30 minutes to ensure complete cell lysis and complete DNA release. (3) Transfer the lysed sample solution obtained in step (2) to a silica membrane adsorption column, place the adsorption column in a collection tube, centrifuge, discard the collection tube and the filtrate; (4) Place the adsorption column in a new collection tube and add a washing buffer containing a high concentration of guanidine salt and anhydrous ethanol to the adsorption column. Centrifuge, discard the collection tube and the filtered waste liquid. Place the adsorption column in a new collection tube and add an elution buffer containing anhydrous ethanol and a low concentration of buffer salt to the adsorption column. Centrifuge, discard the collection tube and the filtered waste liquid. (5) Place the adsorption column obtained in step (4) into a new collection tube, centrifuge for 2-5 minutes to completely dry the membrane of the adsorption column, place the adsorption column into a new collection tube, add sterile ultrapure water or distilled water to the adsorption column, incubate at room temperature for 3-10 minutes, centrifuge for 30-180 seconds, collect the filtrate, and you will get the product.

2. The extraction method according to claim 1, characterized in that, The specific process of step (1) is as follows: Use a microfiber swab or sampling tape pre-moistened with physiological saline to gently and evenly wipe a specific area of ​​the subject's face (including the cheeks and forehead) to collect exfoliated epithelial cells. Immediately place the obtained swab head or tape into the preservation solution, or directly transfer the liquid sample into the preservation solution and mix evenly.

3. The extraction method according to claim 1, characterized in that, The composition of the preservation solution in step (1) is: 5~15mM Tris-HCl, 0.5~1.5 mM EDTA, the balance being water, pH 7.5~8.

5.

4. The extraction method according to claim 1, characterized in that, The lysis buffer in step (2) consists of: 0.5-1.5% SDS, 10-30 mM EDTA, 5-15 mM Tris-HCl, with the balance being water, and pH 7.5-8.5; the binding buffer consists of: 3-5 M guanidine isothiocyanate, 20-40% isopropanol, 0.05-0.5 M Tris-HCl, with the balance being water, and pH 6.0-7.

0.

5. The extraction method according to claim 1, characterized in that, The volume ratio of the sample solution, lysis buffer, binding buffer and anhydrous ethanol in step (2) is 5:1:1:1 to 1:1:1:

1.

6. The extraction method according to claim 1, characterized in that, The final concentration of proteinase K in step (2) is 0.05~0.5 mg / mL.

7. The extraction method according to claim 1, characterized in that, The rotational speed of the oscillation in step (2) is 500~1500 rpm.

8. The extraction method according to claim 1, characterized in that, The silica membrane adsorption column mentioned in step (3) is a QIAamp MinElute column; the centrifugation conditions are: centrifugation at 7500~8500 rpm for 30~180 seconds.

9. The extraction method according to claim 1, characterized in that, The washing buffer in step (4) consists of: 4-5 M guanidine hydrochloride, 20-40 mM Tris-HCl, 80-120 mM NaCl, 50-60% anhydrous ethanol, and the remainder is water; the elution buffer consists of: 5-15 mM Tris-HCl, 0.1-1 mM EDTA, 60-70% anhydrous ethanol, and the remainder is water; the centrifugation conditions are: 13000-15000 rpm for 30-180 seconds.

10. The extraction method according to claim 1, characterized in that, The centrifugation speed in step (5) is 13000~15000 rpm; after step (5) is completed, the obtained filtrate is added back to the adsorption column for a second centrifugation and elution, and the filtrate is collected to obtain the product.

Images