Cracking buffer solution, kit and extraction method for extracting free DNA (Deoxyribose Nucleic Acid) from peripheral blood
By using an SDS-free lysis buffer and the synergistic effect of components such as guanidine hydrochloride, the problems of reduced detection sensitivity and low cfDNA yield caused by SDS residue in existing technologies have been solved, achieving more efficient free DNA extraction.
Patent Information
- Application Number
- CN202511368838.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-23
- Publication Date
- 2025-12-19
AI Technical Summary
Existing lysis buffers often contain SDS when extracting cell-free DNA from peripheral blood, which leads to reduced sensitivity of subsequent detection or false negative results, and low cfDNA yield.
SDS-free lysis buffers were used, including a first lysis buffer and a second lysis buffer, which contained guanidine hydrochloride, ammonium thiocyanate plasma reagent, metal ion chelating agent, trehalose, digestive enzymes and other components, respectively, to extract free DNA through synergistic effects.
It avoids interference from residual SDS, improves detection sensitivity, and significantly increases the amount of free DNA extracted.
Smart Images
Figure SMS_1 
Figure SMS_2 
Figure SMS_3
Abstract
Description
Technical Field
[0001] This invention relates to the field of nucleic acid detection technology, specifically to a lysis buffer, kit, and extraction method for extracting cell-free DNA from peripheral blood. Background Technology
[0002] Cell-free DNA (cfDNA) refers to DNA circulating in blood that exists outside of cells. The vast majority of cfDNA is short-chain and exists at very low concentrations, making it highly susceptible to contamination and dilution by more abundant non-cfDNA. Studies have shown that cfDNA has significant applications in many medical fields, including disease diagnosis, treatment monitoring, and fetal aneuploidy screening. In recent years, with liquid biopsy technology, represented by cfDNA, becoming an important technique for molecular diagnostics or auxiliary diagnostics, cfDNA research has become a hot area in genomic molecular diagnostics research.
[0003] cfDNA extraction generally includes sample lysis, protein precipitation, DNA binding, washing, and final DNA dissolution. However, existing lysis buffers commonly use SDS during the lysis process. Residual SDS may inhibit the activity of enzymes used in subsequent diagnostic procedures or interfere with antibody-antigen binding, leading to reduced sensitivity or false negative results.
[0004] Therefore, it is of great significance to develop SDS-free lysis buffers to extract free DNA without SDS residue. Summary of the Invention
[0005] The lysis buffer and kit for cell-free DNA in peripheral blood provided by this invention do not contain SDS, which can avoid residual SDS in the extraction product and obtain a greater amount of cell-free DNA.
[0006] The present invention achieves the above-mentioned technical objectives through the following technical solutions:
[0007] A lysis buffer for extracting cell-free DNA from peripheral blood, comprising a first lysis buffer and a second lysis buffer;
[0008] The first lysis buffer comprises 3.0-4.0M main lysis buffer, 0.5-1.5M auxiliary lysis buffer, metal ion chelating agent, 0.1-0.5% (w / v) trehalose, 1-5mM dithiothreitol, digestive enzyme, 0.05-0.2% (w / v) sodium dodecyl sarcosinate, 5-10mM borate, and 0.1-0.5mM ascorbic acid; wherein the main lysis buffer comprises guanidine hydrochloride, and the auxiliary lysis buffer comprises at least one of ammonium thiocyanate and guanidine isothiocyanate;
[0009] The second lysis buffer comprises 4.5-5.5M primary lysis buffer, 0.5-1.5M secondary lysis buffer, metal ion chelating agent, 0.1-0.5% (w / v) trehalose, 1-5mM dithiothreitol, digestive enzyme, 0.05-0.2% (w / v) sodium dodecyl sarcosinate, 5-10mM borate, 0.1-0.5mM ascorbic acid, and 0.01-0.05% (w / v) polyethylene glycol; wherein the primary lysis buffer includes guanidine hydrochloride, and the secondary lysis buffer includes urea.
[0010] The lysis buffer for peripheral blood cell-free DNA extraction described above uses proteinase K as the digestive enzyme, and the concentration of proteinase K is 0.5-2.0 mg / mL.
[0011] The lysis buffer for peripheral blood cell-free DNA extraction described above has a metal ion chelating agent concentration of 10-30 mM.
[0012] The lysis buffer for peripheral blood cell-free DNA extraction described above has a pH of 7.5-8.5.
[0013] The lysis buffer for peripheral blood cell-free DNA extraction described above, wherein the borate is selected from one or both of sodium borate and potassium borate.
[0014] The lysis buffer for peripheral blood cell-free DNA extraction described above uses a metal ion chelating agent selected from one or more of ethylenediaminetetraacetic acid (EDTA) or its salts.
[0015] The lysis buffer for peripheral blood cell-free DNA extraction described above includes ammonium thiocyanate and guanidine isothiocyanate in a mass ratio of 1:(1-3).
[0016] The lysis buffer for peripheral blood cell-free DNA extraction described above also includes a buffer solution.
[0017] The lysis buffer for peripheral blood cell-free DNA extraction described above is a Tris-HCl buffer.
[0018] The present invention also provides a kit comprising the above-described lysis buffer.
[0019] The present invention also provides a method for extracting cell-free DNA from peripheral blood, comprising the steps of sequentially digesting and treating peripheral blood with the first lysis buffer and the second lysis buffer to obtain cell-free DNA.
[0020] The lysis buffer provided by this invention does not contain SDS, which can avoid residual SDS in the extraction product, thereby reducing interference with subsequent detection and improving the sensitivity of subsequent detection. Furthermore, through the synergistic effect between the above-mentioned reagents, a greater amount of free DNA is extracted. Detailed Implementation
[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.
[0022] Given the common problem of low cfDNA yield when using existing lysis buffers to lyse peripheral blood to obtain cfDNA, this invention provides a new lysis buffer for peripheral blood cell-free DNA extraction, which includes a first lysis buffer and a second lysis buffer.
[0023] The first lysis buffer comprises 3.0-4.0M main lysis buffer, 0.5-1.5M auxiliary lysis buffer, metal ion chelating agent, 0.1-0.5% (w / v) trehalose, 1-5mM dithiothreitol, digestive enzyme, 0.05-0.2% (w / v) sodium dodecyl sarcosinate, 5-10mM borate, and 0.1-0.5mM ascorbic acid; wherein the main lysis buffer includes guanidine hydrochloride, and the auxiliary lysis buffer includes at least one of ammonium thiocyanate and guanidine isothiocyanate;
[0024] The second lysis buffer includes 4.5-5.5M main lysis buffer, 0.5-1.5M auxiliary lysis buffer, metal ion chelating agent, 0.1-0.5% (w / v) trehalose, 1-5mM dithiothreitol, digestive enzyme, 0.05-0.2% (w / v) sodium dodecyl sarcosinate, 5-10mM borate, 0.1-0.5mM ascorbic acid, and 0.01-0.05% (w / v) polyethylene glycol; wherein the main lysis buffer includes guanidine hydrochloride, and the auxiliary lysis buffer includes urea.
[0025] Using this lysis buffer to treat peripheral blood for cfDNA extraction results in products free of SDS residue, which helps reduce interference with subsequent detection and increases yield.
[0026] Specifically, the concentration of the main dissociating agent in the first lysis buffer can be 3.0M, 3.5M, 4.0M, or any value between any two of the above ranges. The concentration of the main dissociating agent in the second lysis buffer can be 4.5M, 5.0M, 5.5M, or any value between any two of the above ranges.
[0027] The concentration of the auxiliary ionizing agent in the first and second lysis buffers can be 0.5M, 0.8M, 1.2M, 1.5M, or any value between any two of the above ranges.
[0028] The concentration of trehalose (mass-volume fraction) can be 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, or any value between any two of the above ranges.
[0029] The concentration of dithiothreitol can be 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, or any value between any two of the above ranges.
[0030] The mass-volume fraction of sodium dodecyl sarcosinate can be 0.05%, 0.08%, 0.1%, 0.15%, 0.2%, or any value between any two of the above ranges.
[0031] The concentration of borate can be 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, or any value between any two of the above ranges.
[0032] The concentration of ascorbic acid in the first lysis buffer can be 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, or any value between any two of the above ranges.
[0033] The concentration (mass / volume fraction) of polyethylene glycol in the second lysis buffer is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, or any value between any two of the above ranges.
[0034] It should be noted that for components with the same content range in the first and second lysis solutions, their specific contents may be the same or different.
[0035] In the above-described manner, the metal ion chelating agent inhibits nuclease activity by chelating metal ions. In some specific embodiments, its concentration is 10-30 mM. Exemplary values can be 10 mM, 12 mM, 15 mM, 18 mM, 20 mM, 22 mM, 25 mM, 27 mM, 28 mM, 30 mM, or any value between any two of the above ranges.
[0036] Proteinase K can specifically hydrolyze proteins, further removing proteins bound to cfDNA and increasing cfDNA yield. In some specific embodiments, the concentration of proteinase K is 0.5-2.0 mg / mL. Exemplary values can be 0.5 mg / mL, 0.8 mg / mL, 1.2 mg / mL, 1.5 mg / mL, 1.8 mg / mL, 2.0 mg / mL, and any value between any two of the above ranges.
[0037] It is easy to understand that proteinase K is more active at pH 7.5-8.5, therefore, the pH of lysis buffer is usually controlled between 7.5 and 8.5.
[0038] In this invention, the type of borate is not specifically limited. For example, one or two of sodium borate and potassium borate can be selected.
[0039] In this invention, the choice of metal ion chelating agent is not specifically limited. Conventional metal ion chelating agents in the art can be used. For example, the metal ion chelating agent can be one or more of ethylenediaminetetraacetic acid (EDTA) or its salts.
[0040] Further research showed that when ammonium thiocyanate and guanidine isothiocyanate in a mass ratio of 1:(1-3) were used together as auxiliary lysis agents in the first lysis buffer, the lysis effect was better, more DNA was extracted, and the proportion of free DNA was also higher.
[0041] Understandably, a buffer solution is usually added to the system to ensure its stability. The type of buffer solution is not specifically limited. In some specific implementations, the buffer solution is a Tris-HCl buffer, and its pH can be adjusted and maintained at an optimal level using 5-50 mM Tris-HCl buffer.
[0042] The present invention also provides a kit comprising the lysis buffer described above. Because this kit contains the lysis buffer, for the same reason, the extracted cfDNA does not contain residual SDS, thus not affecting the sensitivity of subsequent detection and analysis, and yields a greater amount of free DNA.
[0043] The present invention also provides a method for extracting cell-free DNA from peripheral blood, comprising the steps of sequentially digesting peripheral blood plasma with the first lysis buffer and the second lysis buffer described above to obtain cell-free DNA.
[0044] In this invention, the method of obtaining peripheral blood plasma samples is not limited, and conventional methods in the art can be used. In some specific embodiments, peripheral blood plasma can be obtained by separating it as follows: Human peripheral blood samples are centrifuged at 1400-1800g under mild conditions (e.g., 20°C) for 8-15 minutes. After collecting the cell-free supernatant plasma, it is centrifuged again at 10000-15000g for 8-15 minutes to remove cell debris and other impurities, thus obtaining the peripheral blood plasma sample.
[0045] The plasma cfDNA extraction kit of the present invention will be described in detail below with reference to specific embodiments.
[0046] Unless otherwise specified, the experimental procedures in the following examples are all routine procedures; reagents and materials are commercially available unless otherwise specified.
[0047] Proteinase K was purchased from Beijing TransGen Biotech Co., Ltd., product number GE201-01.
[0048] Example 1
[0049] This embodiment provides a lysis buffer, as detailed below:
[0050]
[0051] Example 2
[0052] This embodiment provides a lysis buffer, as detailed below:
[0053]
[0054] Example 3
[0055] This embodiment provides a lysis buffer, as detailed below:
[0056]
[0057] Example 4
[0058] This embodiment provides a lysis buffer, as detailed below:
[0059]
[0060] Example 5
[0061] This embodiment provides a lysis buffer, as detailed below:
[0062]
[0063] Example 6
[0064] This embodiment provides a lysis buffer, as detailed below:
[0065]
[0066] Comparative Example 1
[0067] This comparative example provides a lysis buffer that differs from Example 2 in that it includes only the first lysis buffer.
[0068] Comparative Example 2
[0069] This comparative example provides a lysis buffer that differs from Example 2 in that it includes only the second lysis buffer.
[0070] Comparative Example 3
[0071] This comparative example provides a lysis buffer that differs from Example 2 in that it does not contain borate, as detailed below:
[0072] Comparative Example 4
[0073] This comparative example provides a lysis buffer, which differs from Example 2 in that glycerol is used to replace trehalose in both the first and second lysis buffers by equal mass, as detailed below:
[0074] Comparative Example 5
[0075] This comparative example provides a lysis buffer that differs from Example 2 in that it does not contain ascorbic acid, as detailed below:
[0076] Experimental Example 1
[0077] Five preserved peripheral blood samples from healthy individuals were collected. After centrifugation at 1600g and 20℃ for 10 minutes, the supernatant cell-free plasma was collected. Then, the samples were centrifuged again at 12000g and 4℃ for 10 minutes to remove cell debris and other impurities, resulting in five plasma samples.
[0078] One mL of the treated plasma was taken and lysed using the lysis buffers from the above examples and comparative examples, as well as a conventional SDS lysis buffer. The conventional SDS lysis buffer consisted of 50 mM Tris-HCl (pH 8.0), 50 mM EDTA, 2% SDS (w / v), 500 mM NaCl, 1% Triton X-100 (w / v), and 20% isopropanol (v / v). The detailed extraction method is as follows:
[0079] Add 1 mL of the first lysis buffer to each 1 mL plasma sample, mix thoroughly, and incubate at 55 °C for 30 minutes. During this period, mix and disperse the sample intermittently to ensure complete lysis. The resulting digested plasma sample is then centrifuged at 10000 g for 10 minutes to remove the precipitate.
[0080] Then, add the second lysis buffer to the supernatant again and repeat the above operation to remove the precipitate and collect the supernatant (this step is not included in Comparative Examples 1 and 2). Add silica-based magnetic beads (from Thermo Fisher Scientific MagMAX™ Cell-Free DNA Separation Kit) and 1 mL of binding buffer to the supernatant, vortex to mix, and incubate at room temperature for 5-10 min. After magnetic separation, wash twice with 75% ethanol to remove proteins and salts, dry the magnetic beads, and elute with 50 μL of sterile water.
[0081] In the above, the composition of the binding solution is: 4M ammonium thiocyanate, 30v / v% isopropanol, and water.
[0082] The yield of the extracted solution was tested, and the concentration was detected by using a Qubit fluorescence quantitative quantitation instrument (Thermo Fisher Scientific). The results are shown in Table 1 below.
[0083] The size distribution of free DNA fragments in the extract was detected using a Qsep100 capillary electrophoresis apparatus with S3 clips. The percentage of fragments with a target fragment of less than 150-200 bp in the total nucleic acid was obtained. The results are shown in Table 2 below.
[0084] Table 1. DNA yield in extracts obtained after treatment with different lysis buffers (in ng).
[0085]
[0086] Table 2. Percentage (%) of fragments shorter than 150-200 bp in the extracts obtained after treatment with different lysis buffers.
[0087]
[0088] The results above show that when plasma samples are lysed and free DNA is extracted using the lysis buffer provided in this embodiment of the invention, the concentration of DNA in the extract is higher, and the proportion of 150-200bp fragments is greater, indicating that the lysis buffer provided in this invention is beneficial to increasing the yield of cfDNA.
[0089] Experimental Example 2
[0090] The cfDNA obtained by lysis and separation using the lysis buffer from Example 2 and conventional SDS lysis buffer in Experiment 1 was used to construct a library. The specific method is as follows:
[0091] End repair: Fragmented cfDNA was incubated at 37°C for 60 minutes in a reaction system containing 1×T4 DNA ligase buffer, 0.3 mM dNTPs (dATP, dGTP, dCTP, dTTP), 3 U / μL T4 DNA polymerase, and 3 U / μL T4 polynucleotide kinase.
[0092] Adapter ligation: The end-repaired DNA fragment was mixed with an appropriate amount of sequencing adapter (TruSeq adapter, 77 bp in length) and incubated at 20°C for 2 hours in a reaction system containing 1×T4 DNA ligase buffer and 3 U / μL T4 DNA ligase.
[0093] PCR amplification: PCR was performed in a reaction system containing 1×PCR buffer, 0.3 mM dNTPs, 1 μM upstream primer, 1 μM downstream primer, 3 U / μL DNA polymerase, and an appropriate amount of template DNA (ligation product). The reaction conditions were: 95°C pre-denaturation for 4 minutes; followed by 7 cycles of amplification, each cycle consisting of 95°C denaturation for 20 seconds, 60°C annealing for 20 seconds, 72°C extension for 40 seconds, and a final extension at 72°C for 10 minutes.
[0094] Library purification: PCR amplification products were purified using gel electrophoresis.
[0095] Library quantification and quality control: The purified library was quantitatively analyzed using a Qubit real-time fluorescence analyzer, and the size of the library fragments was detected by a Qsep100 capillary electrophoresis instrument to determine the concentration and fragment size (cfDNA + adapter length is about 320bp) distribution of the library. The detection results are shown in Tables 3 and 4 below.
[0096] Table 3 shows the mass concentration of DNA molecules in the library (unit: ng / μL).
[0097]
[0098] Table 4 shows the percentage of the target fragment (275-400bp) in the library (unit: %).
[0099]
[0100] The results above show that the DNA extracted using the extraction kit in Example 2 yielded a higher library output when constructed under the same conditions, further demonstrating that the lysis buffer provided by this invention is beneficial for increasing the yield of cfDNA extraction.
[0101] 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 lysis buffer for extracting cell-free DNA from peripheral blood, characterized in that, Includes a first lysis buffer and a second lysis buffer; The first lysis buffer comprises 3.0-4.0M main lysis buffer, 0.5-1.5M auxiliary lysis buffer, metal ion chelating agent, 0.1-0.5% (w / v) trehalose, 1-5mM dithiothreitol, digestive enzyme, 0.05-0.2% (w / v) sodium dodecyl sarcosinate, 5-10mM borate, and 0.1-0.5mM ascorbic acid; wherein the main lysis buffer comprises guanidine hydrochloride, and the auxiliary lysis buffer comprises at least one of ammonium thiocyanate and guanidine isothiocyanate; The second lysis buffer comprises 4.5-5.5 M primary lysis buffer, 0.5-1.5 M secondary lysis buffer, metal ion chelating agent, 0.1-0.5% (w / v) trehalose, 1-5 mM dithiothreitol, digestive enzyme, 0.05-0.2% (w / v) sodium dodecyl sarcosinate, 5-10 mM borate, 0.1-0.5 mM ascorbic acid, and 0.01-0.05% (w / v) polyethylene glycol; wherein the primary lysis buffer includes guanidine hydrochloride, and the secondary lysis buffer includes urea.
2. The lysis buffer for peripheral blood cell-free DNA extraction according to claim 1, characterized in that, The digestive enzyme is proteinase K, and the concentration of proteinase K is 0.5-2.0 mg / mL.
3. The lysis buffer for peripheral blood cell-free DNA extraction according to claim 1 or 2, characterized in that, The concentration of the metal ion chelating agent is 10-30 mM; and / or The pH of the lysis buffer is 7.5-8.
5.
4. The lysis buffer for peripheral blood cell-free DNA extraction according to claim 1 or 2, characterized in that, The borate is selected from one or two of sodium borate and potassium borate.
5. The lysis buffer for peripheral blood cell-free DNA extraction according to claim 1 or 2, characterized in that, The metal ion chelating agent is selected from one or more of ethylenediaminetetraacetic acid or its salts.
6. The lysis buffer for peripheral blood cell-free DNA extraction according to claim 5, characterized in that, The auxiliary liquid release agent includes ammonium thiocyanate and guanidine isothiocyanate, and the mass ratio of the two is 1:(1-3).
7. The lysis buffer for peripheral blood cell-free DNA extraction according to claim 1 or 2, characterized in that, It also includes buffer solutions.
8. The lysis buffer for peripheral blood cell-free DNA extraction according to claim 7, characterized in that, The buffer solution is Tris-HCl buffer.
9. A reagent kit, characterized in that, Includes the lysis buffer according to any one of claims 1-8.
10. A method for extracting cell-free DNA from peripheral blood, characterized in that, The method includes the step of sequentially digesting peripheral blood plasma with the first lysis buffer and the second lysis buffer as described in any one of claims 1-8 to obtain cell-free DNA.