Extraction method of trace blood DNA

Abstract

The invention relates to the technical field of biomedicine, in particular to a trace blood DNA (deoxyribonucleic acid) extraction method which comprises the following steps: S1, mixing a blood sample and lysate according to a ratio of 1: 3, uniformly mixing by vortex, and centrifuging; s2, protease K is added, vortex mixing is carried out, incubation is carried out at 55 DEG C for 15 min, centrifugation is carried out for 30 s, and 500 microliters of a binding solution is added; and S3, completely transferring the liquid into a purification column with a collection tube, and standing for 2 minutes. The extraction method of the trace blood DNA comprises the following steps: denaturating protein through SDS (Sodium Dodecyl Sulfate), and separating nucleic acid; the binding liquid adopts high salt and low PH, so that the long fragment nucleic acid can be more easily bound on the adsorption column, guanidine hydrochloride can enhance the splitting effect, and the nucleic acid in the blood sample can be fully released due to the dual splitting effect; and on the other hand, the key point is that the step of removing red blood cells by red blood cell lysate is avoided, but the whole blood is directly lysed, so that the loss of white blood cells is reduced, the operation steps are simplified, and the method is suitable for extracting fresh and frozen anticoagulant blood samples.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically a method for extracting trace amounts of blood DNA. Background Technology

[0002] In the biomedical field, blood DNA extraction technology is an important research and application tool, used for the diagnosis and treatment of various diseases, as well as for gene analysis and detection in basic research. Current blood DNA extraction technologies primarily employ chemical methods, such as using enzymes like proteinase K to break down cell membranes, followed by the extraction of DNA using organic solvents (such as phenol and chloroform). Clinical blood samples are a valuable resource for medical research, and their availability is extremely limited, often in the micro-scale. The ability to extract DNA from minute amounts of blood samples not only conserves precious blood resources but also reduces the difficulty of collecting clinical blood samples for medical research.

[0003] Currently, blood DNA extraction methods are broadly classified into three categories: phenol-chloroform extraction, centrifugation column extraction, and magnetic bead extraction. Phenol-chloroform extraction is relatively inexpensive, but it uses toxic chemical reagents and is complex and time-consuming, making it unsuitable for large-scale sample processing. Centrifugation column extraction and magnetic column extraction are commonly used blood DNA extraction methods, but these methods are typically cumbersome, and some require at least 200 μL of blood sample for DNA extraction (e.g., CN202111542009.1, CN201310593112.8), failing to meet the needs of extracting DNA from small amounts of blood.

[0004] To address the aforementioned issues, we propose an improvement method for extracting trace amounts of blood DNA. Summary of the Invention

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0006] This invention provides a method for extracting trace amounts of blood DNA, comprising the following steps:

[0007] S1: Mix the blood sample with the lysis buffer at a ratio of 1:3, vortex to mix, and centrifuge;

[0008] S2: Add proteinase K, vortex to mix, incubate at 55℃ for 15 min, centrifuge for 30 s, and add 500 μL of binding solution;

[0009] S3: Transfer all the liquid to the purification column containing the collection tube, let it stand for 2 minutes, centrifuge, and discard the waste liquid in the collection tube;

[0010] S4: Add washing solution to the purification column and wash, then add rinsing solution to the purification column and rinse; repeat step S4 once, then centrifuge and air dry the purification column.

[0011] S5: Add elution buffer TE to the purification column and elute. Collect the DNA solution in the centrifuge tube.

[0012] As a preferred embodiment of the present invention, the blood sample in step S1 is fresh blood or frozen anticoagulated blood, and the sample volume is 50 μL.

[0013] As a preferred embodiment of the present invention, the lysis solution in step S1 contains ammonium chloride, SDS, sodium acetate and NaCl.

[0014] As a preferred embodiment of the present invention, the binding solution in step S2 comprises guanidine hydrochloride, Tween 20 and anhydrous ethanol.

[0015] As a preferred embodiment of the present invention, the washing solution in step S4 comprises guanidine isothiocyanate, Tris-HCl (pH 8.0), EDTA (pH 8.0) and anhydrous ethanol;

[0016] The rinsing solution in step S4 contains Tris-HCl (pH 8.0) and EDTA (pH 8.0).

[0017] As a preferred embodiment of the present invention, the eluent in step S4 is a TE buffer containing Tris-HCl (pH 8.0) and EDTA (pH 8.0).

[0018] As a preferred embodiment of the present invention, the centrifugation in step S1 is for 30 seconds; the centrifugation conditions in steps S3 and S4 are 12000 rpm for 30 seconds; and the centrifugation conditions after elution in step S5 are 12000 rpm for 2 minutes.

[0019] As a preferred embodiment of the present invention, the purification column is a silica membrane adsorption column, used to bind DNA under high salt and low pH conditions.

[0020] The beneficial effects of this invention are as follows: This method for extracting trace amounts of blood DNA involves denaturing proteins and separating nucleic acids using SDS; the binding solution employs a high-salt, low-pH method to facilitate the binding of long-fragment nucleic acids onto the adsorption column, while guanidine hydrochloride enhances the lysis effect. This dual lysis effect fully releases the nucleic acids in the blood sample. Crucially, this invention eliminates the step of removing red blood cells using red blood cell lysis buffer, instead directly lysing whole blood. This reduces white blood cell loss and simplifies the operation, making it suitable for extracting both fresh and frozen anticoagulated blood samples. Attached Figure Description

[0021] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0022] Figure 1 This is a schematic flowchart of a method for extracting trace amounts of blood DNA according to the present invention; Detailed Implementation

[0023] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0024] Example 1: As Figure 1 As shown, a method for extracting trace amounts of blood DNA includes the following steps:

[0025] S1: Mix the blood sample with the lysis buffer at a ratio of 1:3, vortex to mix, and centrifuge;

[0026] S2: Add proteinase K, vortex to mix, incubate at 55℃ for 15 min, centrifuge for 30 s, and add 500 μL of binding solution;

[0027] S3: Transfer all the liquid to the purification column containing the collection tube, let it stand for 2 minutes, centrifuge, and discard the waste liquid in the collection tube;

[0028] S4: Add washing solution to the purification column and wash, then add rinsing solution to the purification column and rinse; repeat step S4 once, then centrifuge and air dry the purification column.

[0029] S5: Add elution buffer TE to the purification column and elute. Collect the DNA solution in the centrifuge tube.

[0030]

[0031] The table above shows the formulations of the lysis buffer, binding buffer, washing buffer, rinsing buffer, and elution buffer.

[0032] The blood sample in step S1 is either fresh blood or frozen anticoagulated blood, and the sample volume is 50 μL.

[0033] The lysis solution in step S1 contains ammonium chloride, SDS, sodium acetate, and NaCl.

[0034] The binding solution in step S2 contains guanidine hydrochloride, Tween 20, and anhydrous ethanol.

[0035] The washing solution in step S4 contains guanidine isothiocyanate, Tris-HCl (pH 8.0), EDTA (pH 8.0), and anhydrous ethanol;

[0036] The rinsing solution in step S4 contains Tris-HCl (pH 8.0) and EDTA (pH 8.0).

[0037] The elution buffer in step S4 is TE buffer, which contains Tris-HCl (pH 8.0) and EDTA (pH 8.0).

[0038] In step S1, the centrifugation time is 30 seconds; in steps S3 and S4, the centrifugation conditions are 12000 rpm for 30 seconds; in step S5, the centrifugation conditions after elution are 12000 rpm for 2 minutes.

[0039] The purification column is a silica membrane adsorption column, used to bind DNA under high salt and low pH conditions.

[0040]

[0041] The table above shows the formulations of the lysis buffer and binding buffer, specifically:

[0042] Prepare a small blood sample. Collect 50 μL of blood sample frozen at -80°C or a fresh blood sample.

[0043] Lysis: Add 50 μL of blood sample and 150 μL of lysis buffer to a 1.5 mL centrifuge tube, vortex for 5 min, and centrifuge for 30 s;

[0044] Binding: Add 20 μL of proteinase K. Vortex to mix, incubate in a metal bath at 55 °C for 15 min; centrifuge for 30 s, add 500 μL of binding solution to the centrifuge tube, mix well, and centrifuge for 30 s; 4. Purification column purification: Transfer all liquid to a purification column containing a collection tube, let stand for 2 min, centrifuge at 12000 rpm (~13,400×g) for 30 s, and discard the waste liquid in the collection tube;

[0045] Washing: Add 500 μL of washing buffer to the purification column, centrifuge at 12000 rpm (~13,400×g) for 30 s, and discard the waste liquid in the collection tube; add 600 μL of rinsing buffer to the purification column, centrifuge at 12000 rpm (~13,400×g) for 30 s, and discard the waste liquid in the collection tube; repeat step 6 once, and then centrifuge at 12000 rpm (~13,400×g) for 2 min;

[0046] Air dry: Transfer the purification column to a clean 1.5mL centrifuge tube, open the cap, and air dry for 2 minutes;

[0047] Elution: Add 100 μL of elution buffer TE to the purification column, let stand for 2 min, then centrifuge at 12000 rpm (~13,400×g) for 2 min and collect the DNA solution in the centrifuge tube.

[0048] Example 2:

[0049]

[0050] The table above shows the formulations of the lysis buffer and binding solution. Specifically:

[0051] Prepare a small blood sample. Collect 50 μL of blood sample frozen at -80°C or a fresh blood sample.

[0052] Lysis: Add 50 μL of blood sample and 150 μL of lysis buffer to a 1.5 mL centrifuge tube and vortex for 5 min.

[0053] Binding: Centrifuge for 30 seconds, add 20 μL of proteinase K. Vortex to mix, incubate in a metal bath at 55°C for 15 minutes; centrifuge for 30 seconds, add 500 μL of binding solution to the centrifuge tube;

[0054] Purification column purification: After mixing, centrifuge for 30s, transfer all the liquid to the purification column containing the collection tube, let stand for 2min, centrifuge at 12000rpm (~13,400×g) for 30s, and discard the waste liquid in the collection tube.

[0055] Washing: Add 500 μL of washing buffer to the purification column, centrifuge at 12000 rpm (~13,400×g) for 30 s, and discard the waste liquid in the collection tube; add 600 μL of rinsing buffer to the purification column, centrifuge at 12000 rpm (~13,400×g) for 30 s, and discard the waste liquid in the collection tube; repeat step 6 once, and then centrifuge at 12000 rpm (~13,400×g) for 2 min;

[0056] Air dry: Transfer the purification column to a clean 1.5mL centrifuge tube, open the cap, and air dry for 2 minutes;

[0057] Elution: Add 100 μL of elution buffer TE to the purification column, let stand for 2 min, then centrifuge at 12000 rpm (~13,400×g) for 2 min and collect the DNA solution in the centrifuge tube.

[0058] Example 3:

[0059]

[0060] The table above shows the formulations of the lysis buffer and binding solution. Specifically:

[0061] Prepare a small blood sample. Collect 50 μL of blood sample frozen at -80°C or a fresh blood sample.

[0062] Lysis: Add 50 μL of blood sample and 150 μL of lysis buffer to a 1.5 mL centrifuge tube and vortex for 5 min.

[0063] Binding: Centrifuge for 30 seconds, add 20 μL of proteinase K. Vortex to mix, incubate in a metal bath at 55°C for 15 minutes; centrifuge for 30 seconds, add 500 μL of binding solution to the centrifuge tube;

[0064] Purification column purification: After mixing, centrifuge for 30s, transfer all the liquid to the purification column containing the collection tube, let stand for 2min, centrifuge at 12000rpm (~13,400×g) for 30s, and discard the waste liquid in the collection tube.

[0065] Washing: Add 500 μL of washing buffer to the purification column, centrifuge at 12000 rpm (~13,400×g) for 30 s, and discard the waste liquid in the collection tube; add 600 μL of rinsing buffer to the purification column, centrifuge at 12000 rpm (~13,400×g) for 30 s, and discard the waste liquid in the collection tube; repeat step 6 once, and then centrifuge at 12000 rpm (~13,400×g) for 2 min;

[0066] Air dry: Transfer the purification column to a clean 1.5mL centrifuge tube, open the cap, and air dry for 2 minutes;

[0067] Elution: Add 100 μL of elution buffer TE to the purification column, let stand for 2 min, then centrifuge at 12000 rpm (~13,400×g) for 2 min and collect the DNA solution in the centrifuge tube.

[0068] Example 4:

[0069]

[0070] The table above shows the formulations of the lysis buffer and binding solution. Specifically:

[0071] Prepare a small blood sample. Collect 50 μL of blood sample frozen at -80°C or a fresh blood sample.

[0072] Lysis: Add 50 μL of blood sample and 150 μL of lysis buffer to a 1.5 mL centrifuge tube and vortex for 5 min.

[0073] Binding: Centrifuge for 30 seconds, add 20 μL of proteinase K. Vortex to mix, incubate in a metal bath at 55°C for 15 minutes; centrifuge for 30 seconds, add 500 μL of binding solution to the centrifuge tube;

[0074] Purification column purification: After mixing, centrifuge for 30s, transfer all the liquid to the purification column containing the collection tube, let stand for 2min, centrifuge at 12000rpm (~13,400×g) for 30s, and discard the waste liquid in the collection tube.

[0075] Washing: Add 500 μL of washing buffer to the purification column, centrifuge at 12000 rpm (~13,400×g) for 30 s, and discard the waste liquid in the collection tube; add 600 μL of rinsing buffer to the purification column, centrifuge at 12000 rpm (~13,400×g) for 30 s, and discard the waste liquid in the collection tube; repeat step 6 once, and then centrifuge at 12000 rpm (~13,400×g) for 2 min;

[0076] Air dry: Transfer the purification column to a clean 1.5mL centrifuge tube, open the cap, and air dry for 2 minutes;

[0077] Elution: Add 100 μL of elution buffer TE to the purification column, let stand for 2 min, then centrifuge at 12000 rpm (~13,400×g) for 2 min and collect the DNA solution in the centrifuge tube.

[0078] Example 5:

[0079]

[0080]

[0081] The table above shows the formulations of the lysis buffer and binding solution. Specifically:

[0082] Prepare a small blood sample. Collect 50 μL of blood sample frozen at -80°C or a fresh blood sample.

[0083] Lysis: Add 50 μL of blood sample and 150 μL of lysis buffer to a 1.5 mL centrifuge tube and vortex for 5 min.

[0084] Binding: Centrifuge for 30 seconds, add 20 μL of proteinase K. Vortex to mix, incubate in a metal bath at 55°C for 15 minutes; centrifuge for 30 seconds, add 500 μL of binding solution to the centrifuge tube;

[0085] Purification column purification: After mixing, centrifuge for 30s, transfer all the liquid to the purification column containing the collection tube, let stand for 2min, centrifuge at 12000rpm (~13,400×g) for 30s, and discard the waste liquid in the collection tube.

[0086] Washing: Add 500 μL of washing buffer to the purification column, centrifuge at 12000 rpm (~13,400×g) for 30 s, and discard the waste liquid in the collection tube; add 600 μL of rinsing buffer to the purification column, centrifuge at 12000 rpm (~13,400×g) for 30 s, and discard the waste liquid in the collection tube; repeat step 6 once, and then centrifuge at 12000 rpm (~13,400×g) for 2 min;

[0087] Air dry: Transfer the purification column to a clean 1.5mL centrifuge tube, open the cap, and air dry for 2 minutes;

[0088] Elution: Add 100 μL of elution buffer TE to the purification column, let stand for 2 min, then centrifuge at 12000 rpm (~13,400×g) for 2 min and collect the DNA solution in the centrifuge tube.

[0089] Example 6:

[0090]

[0091]

[0092] The table above shows the formulations of the lysis buffer and binding solution. Specifically:

[0093] Prepare a small blood sample. Collect 50 μL of blood sample frozen at -80°C or a fresh blood sample.

[0094] Lysis: Add 50 μL of blood sample and 150 μL of lysis buffer to a 1.5 mL centrifuge tube, vortex for 5 min; centrifuge for 30 s, add 20 μL of proteinase K. Vortex and incubate in a metal bath at 55 °C for 15 min;

[0095] Binding: Centrifuge for 30 seconds, then add 500 μL of binding solution to the centrifuge tube;

[0096] Purification column purification: After mixing, centrifuge for 30s, transfer all the liquid to the purification column containing the collection tube, let stand for 2min, centrifuge at 12000rpm (~13,400×g) for 30s, and discard the waste liquid in the collection tube.

[0097] Washing: Add 500 μL of washing buffer to the purification column, centrifuge at 12000 rpm (~13,400×g) for 30 s, and discard the waste liquid in the collection tube; add 600 μL of rinsing buffer to the purification column, centrifuge at 12000 rpm (~13,400×g) for 30 s, and discard the waste liquid in the collection tube; repeat step 6 once, and then centrifuge at 12000 rpm (~13,400×g) for 2 min;

[0098] Air dry: Transfer the purification column to a clean 1.5mL centrifuge tube, open the cap, and air dry for 2 minutes;

[0099] Elution: Add 100 μL of elution buffer TE to the purification column, let stand for 2 min, then centrifuge at 12000 rpm (~13,400×g) for 2 min and collect the DNA solution in the centrifuge tube.

[0100] Comparative Example 1:

[0101] Prepare a small blood sample. Collect 50 μL of blood sample frozen at -80°C or a fresh blood sample.

[0102] DNA was extracted using the blood / cell / tissue genomic DNA extraction kit sold by Tiangen Biotech (Beijing) Co., Ltd. Specific operating procedures are detailed in the kit's instruction manual.

[0103] Comparative Example 2:

[0104] Prepare a small blood sample. Collect 100 μL of blood sample frozen at -80°C or a fresh blood sample.

[0105] DNA was extracted using the blood / cell / tissue genomic DNA extraction kit sold by Tiangen Biotech (Beijing) Co., Ltd. Specific operating procedures are detailed in the kit's instruction manual.

[0106] The concentration of nucleic acids extracted in each example was tested using NANODROPONE, and the test results are shown in the table below:

[0107]

[0108]

[0109] The results are shown in the table above. It is clear that the DNA content extracted in Examples 1-5 is higher than that extracted in Comparative Examples 1-2. The DNA content extracted in Examples 1-5 reaches over 30 ng per 100 μL. The sample in Example 3 has the highest nucleic acid content, while the sample in Example 2 has a slightly lower nucleic acid content. However, overall, the nucleic acid content extracted from each example is above 30 ng, reaching a maximum of 34 ng. In Comparative Example 1, using 50 μL of trace blood for DNA extraction resulted in poor extraction quality, less than 20 μg. In Comparative Example 2, increasing the blood sample volume to 100 μL slightly improved the DNA content, but it was still lower than the DNA content extracted in Examples 1-5 of this invention. The results of these examples demonstrate that the improved formulation of this invention can significantly yield higher DNA content. Compared to widely used blood DNA extraction kits on the market, the trace blood DNA extraction method of this invention is more suitable for extracting trace blood DNA, allowing the sample volume to be reduced to 50 μL while still achieving good extraction results and obtaining higher DNA content.

[0110] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for extracting trace amounts of blood DNA, characterized in that, Includes the following steps: S1: Mix the blood sample with the lysis buffer at a ratio of 1:3, vortex to mix, and centrifuge; S2: Add proteinase K, vortex to mix, incubate at 55℃ for 15 min, centrifuge for 30 s, and add 500 μL of binding solution; S3: Transfer all the liquid to the purification column containing the collection tube, let it stand for 2 minutes, centrifuge, and discard the waste liquid in the collection tube; S4: Add washing solution to the purification column and wash, then add rinsing solution to the purification column and rinse; repeat step S4 once, then centrifuge and air dry the purification column. S5: Add elution buffer TE to the purification column and elute. Collect the DNA solution in the centrifuge tube.

2. The method for extracting trace amounts of blood DNA according to claim 1, characterized in that, The blood sample in step S1 is fresh blood or frozen anticoagulated blood, and the sample volume is 50 μL.

3. The method for extracting trace amounts of blood DNA according to claim 1, characterized in that, The lysis solution in step S1 contains ammonium chloride, SDS, sodium acetate, and NaCl.

4. The method for extracting trace amounts of blood DNA according to claim 1, characterized in that, The binding solution in step S2 comprises guanidine hydrochloride, Tween 20, and anhydrous ethanol.

5. The method for extracting trace amounts of blood DNA according to claim 1, characterized in that, The washing solution in step S4 contains guanidine isothiocyanate, Tris-HCl (pH 8.0), EDTA (pH 8.0), and anhydrous ethanol; The rinsing solution in step S4 contains Tris-HCl (pH 8.0) and EDTA (pH 8.0).

6. The method for extracting trace amounts of blood DNA according to claim 1, characterized in that, The elution buffer in step S4 is a TE buffer containing Tris-HCl (pH 8.0) and EDTA (pH 8.0).

7. The method for extracting trace amounts of blood DNA according to claim 1, characterized in that, The centrifugation in step S1 is for 30 seconds; the centrifugation conditions in steps S3 and S4 are 12000 rpm for 30 seconds; and the centrifugation conditions after elution in step S5 are 12000 rpm for 2 minutes.

8. The method for extracting trace amounts of blood DNA according to claim 1, characterized in that, The purification column is a silica membrane adsorption column, used to bind DNA under high salt and low pH conditions.

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