A reagent system and method for extracting genomic DNA from complex contaminated biological samples under multiple conditions

Abstract

The application belongs to the field of analysis and detection, and relates to a reagent system and an extraction method for extracting genomic DNA of complex contaminated biological samples suitable for multiple working conditions, comprising a lysis solution, a binding solution, a rinsing solution and an elution solution; wherein the lysis solution is composed of the following raw materials: 20-40 mmol / L Tris-HCl, 50-100 mmol / L quaternary ammonium salt complex system, 15-30 mmol / L lauryl amide surfactant, 2%-3% (volume fraction) Triton X-100 and 10-15 mmol / L EDTA, and the pH value is 7.4-7.8. A stable buffer control environment is constructed, and the DNA extraction quality of complex contaminated samples is improved. The components of the whole reagent system synergistically act, the overall buffer system is stable, the impurity removal effect is excellent, a variety of solid carriers and different types of extraction equipment can be compatible, and the batch extraction demand of forensic difficult samples under different working conditions can be met.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid extraction technology, specifically relating to a forensic genomic DNA extraction reagent system and extraction method adaptable to multiple working conditions. Background Technology

[0002] In forensic evidence examination, biological contact traces are easily contaminated or embedded by substances such as dust, pigments, grease, and rust. The sample matrix is ​​complex and contains a large number of amplification inhibitors. Currently, commonly used nucleic acid extraction systems often employ guanidine salts as core components; these substances can affect the system's original pH environment and disrupt the stability of the buffer system.

[0003] Currently, to maintain acid-base balance, the amount of buffer salt added is usually increased, but this can easily lead to a decrease in the purity of the final nucleic acid product. If the pH difference between the lysis buffer and the binding buffer is adjusted, it will cause drastic changes in the pH of the system, which will not only cause DNA strand breakage damage, but also aggravate the non-specific adsorption of impurities such as proteins and polysaccharides on the surface of the solid-phase carrier. Ultimately, this will result in low DNA extraction yield and poor STR typing results, making it difficult to meet the detection requirements of complex and difficult samples. Summary of the Invention

[0004] To address the aforementioned issues, this invention provides a forensic genomic DNA extraction reagent system and method adaptable to various working conditions. It constructs a stable buffer and regulation environment, improving the DNA extraction quality of complex contaminated samples. The components of the entire reagent system work synergistically, resulting in a stable overall buffer system, excellent impurity removal, and compatibility with various solid-phase carriers and different types of extraction equipment. This allows it to meet the batch extraction needs of complex forensic samples under different working conditions.

[0005] To achieve the above technical objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a reagent system for extracting genomic DNA from complex contaminated biological samples that is adaptable to various working conditions, comprising: a lysis buffer, a binding buffer, a rinsing buffer, and an elution buffer; The lysis buffer is composed of the following raw materials: 20-40 mmol / L Tris-HCl, 50-100 mmol / L tetraalkyl quaternary ammonium salt complex system, 15-30 mmol / L lauramide surfactant, 2%-3% Triton X-100 (volume fraction), 10-15 mmol / L EDTA, and pH 7.4-7.8.

[0006] A second aspect of the present invention provides an extraction method for a forensic genomic DNA extraction reagent system adapted to multiple working conditions, comprising: Mix the sample to be tested, lysis buffer and proteinase K thoroughly, perform lysis treatment, centrifuge after lysis, and collect the supernatant. Add the binding solution to the supernatant, mix well, then add the solid support, and shake to allow the DNA to bind and accumulate on the surface of the solid support, thus obtaining a DNA-loaded solid support. The solid support was fractionated, rinsed, and eluted to obtain the extract.

[0007] A third aspect of the present invention provides the application of the above-described reagent system in forensic genomic DNA extraction, wherein the sample includes at least one of the following: dust, pigment, grease, rust contamination or embedded contact trace samples, and embedded sample samples.

[0008] The beneficial effects of this invention are as follows: (1) This invention uses a tetraalkyl quaternary ammonium salt complex system as the main salt system. The tetraalkyl quaternary ammonium salt is chemically stable and will not consume the buffer components in the system. After the alkaline lysis buffer is mixed with the weakly alkaline binding buffer, there is no need to increase the amount of buffer salt or increase the pH difference, and the environment can be smoothly transformed into a suitable environment for nucleic acid binding, thereby achieving self-release acid regulation. The addition of PEG8000 to the binding buffer can further optimize the DNA binding effect and improve the nucleic acid enrichment efficiency.

[0009] (2) This invention employs a gradual pH transition method, completely avoiding DNA strand breaks caused by drastic acid-base fluctuations, and effectively ensuring the integrity of long-fragment nucleic acids. The multi-stage rinsing solution, combined with specific concentrations of PEG, sodium chloride, MOPS, and ethanol, can sequentially remove different types of impurities such as proteins, polysaccharides, residual surfactants, and salts, reducing the adhesion of impurities such as pigments, oils, and rust to the solid-phase carrier from the source, and significantly improving the purity and recovery rate of DNA products.

[0010] (3) The overall system of the present invention does not need to rely on high concentration buffer salt to maintain acid-base balance, which reduces the difficulty of subsequent rinsing and impurity removal. It can be used with various solid carriers such as magnetic beads and silicon beads. At the same time, it can be flexibly adapted to different operation modes such as manual operation, semi-automatic equipment, and fully automatic equipment. It has a wide range of applications and good repeatability of test results.

[0011] (4) The method of the present invention is simple, easy to operate, highly practical, and easy to promote. Attached Figure Description

[0012] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0013] Figure 1 STR typing profiles were obtained by processing contaminated contact trace samples using the system described in Example 1 of this invention.

[0014] Figure 2STR typing profiles were obtained by processing the same samples using a control group system.

[0015] Figure 3 Parallel experiments were conducted using the STR typing profiles obtained from contaminated contact trace samples processed with the system described in Example 1 of this invention.

[0016] Figure 4 Parallel experiments used a control group system to process the STR typing profiles obtained from contaminated contact trace samples. Detailed Implementation

[0017] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. The components of the embodiments of the present invention described and shown herein can generally be arranged and designed in various different configurations. Therefore, the following detailed description of the provided embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0019] Terminology Explanation: In this invention, "self-releasing acid regulation" means that after the alkaline lysis buffer is mixed with the weakly alkaline binding buffer, the environment can be smoothly transformed into a suitable environment for nucleic acid binding without the need to increase the amount of buffer salt or increase the acid-base difference.

[0020] As described in the background section, existing nucleic acid extraction systems are not effective for extracting complex contaminated biological samples under various operating conditions. Therefore, this invention provides a genomic DNA extraction reagent system adapted to complex contaminated biological samples under various operating conditions, comprising: a lysis buffer, a binding buffer, a rinsing buffer, and an elution buffer; The lysis buffer is composed of the following raw materials: 20-40 mmol / L Tris-HCl, 50-100 mmol / L tetraalkyl quaternary ammonium salt complex system, 15-30 mmol / L lauramide surfactant, 2%-3% Triton X-100 (volume fraction), 10-15 mmol / L EDTA, and pH 7.4-7.8.

[0021] It should be noted that the present invention does not impose any limiting restrictions on the length of the alkyl carbon chain, nor does it distinguish between long-chain quaternary ammonium salts and short-chain quaternary ammonium salts; the long-chain quaternary ammonium salts such as hexadecyltrimethylammonium bromide and dodecyltrimethylammonium bromide listed below are merely preferred embodiments of the present invention and should not be used to narrow the protection scope of the claims.

[0022] This tetraalkyl quaternary ammonium salt is defined by its chemical structure: The general formula is: The nitrogen atom bonded with four alkyl substituents falls within the scope of this protection, covering C1~C4 symmetrical short-chain quaternary ammonium salts (tetramethyl quaternary ammonium salt, tetraethyl quaternary ammonium salt, tetrabutyl quaternary ammonium salt) as well as alkyl quaternary ammonium salts with longer carbon chains. The combination of various types of tetraalkyl quaternary ammonium salts with different chain lengths is also included in the scope of protection. The type of tetraalkyl quaternary ammonium salt affects its adsorption effect on DNA and its keratin cleavage effect. For example, single-chain trimethyl quaternary ammonium salts have insufficient keratin penetration and low release of hair and nail DNA, while double-chain dimethyl quaternary ammonium salts have weak electrostatic binding ability to DNA and severe nucleic acid loss during the adsorption stage. Therefore, this invention studies the types of tetraalkyl quaternary ammonium salts. Preferably, the tetraalkyl quaternary ammonium salt complex system is selected from at least one of hexadecyltrimethylammonium bromide, dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, and decyldimethylammonium chloride; more preferably, it is hexadecyltrimethylammonium bromide and decyldimethylammonium chloride. Using single-chain trimethyl quaternary ammonium salts and double-chain dimethyl quaternary ammonium salts can better improve the keratin cleavage ability and DNA adsorption ability. At the same time, it is stable when combined with laurylamide surfactants.

[0023] To achieve superior protein dissociation and inhibition of protein-quaternary ammonium salt co-precipitation when surfactants are combined with quaternary ammonium salts, this invention investigated the types of surfactants used. First, anionic and amphoteric surfactants (e.g., SDS, betaine) that would conflict with quaternary ammonium salt cations were ruled out. Then, the effects of combining surfactants with different functional groups with tetraalkyl quaternary ammonium salts were studied and explored over a long period. Preferably, the lauramide surfactant is lauramide monoethanolamine. The long alkyl chain of the quaternary ammonium salt inserts into the hydrophobic region of the α-helix of keratin; lauramide monoethanolamine interpenetrates the gaps in the disulfide bonds of the protein crosslinking, significantly increasing DNA release; lauramide monoethanolamine can encapsulate the hydrophobic residues of denatured proteins, shielding the negative potential points of the proteins and preventing the quaternary ammonium salt cations from forming ionic bonds with the proteins; the protein impurity content in the lysate supernatant is significantly reduced, decreasing subsequent PCR inhibition.

[0024] The composition and amount of each substance in the binding solution affect its binding efficiency with DNA. Therefore, this invention studies the composition of the binding solution. Preferably, the binding solution consists of the following raw materials: a 0.5–1.0 mol / L tetraalkyl quaternary ammonium salt complex system, 10%–15% PEG8000 (mass fraction), 8%–15% alcohols (volume fraction), and a 20–50 mmol / L succinic acid complex buffer component, with a pH value of 7.2–7.3. Relying on the high concentration of the complex quaternary ammonium salt electrostatically binding DNA, it efficiently captures trace amounts of genomic DNA while reducing the risk of co-precipitation of dust, humic pigments, and nucleic acids, thus improving the nucleic acid recovery rate of trace evidence. The binding solution system of this invention, together with the pre-lysis buffer Tris, forms a two-stage pH linkage regulation, compatible with various forensic samples, including hair, nails, old bones, decaying tissue, and traces of dust / rust / grease contamination, significantly improving DNA purity.

[0025] Preferably, the alcohol is ethanol or isopropanol; PEG8000 is combined with ethanol / isopropanol to gently dehydrate and enrich DNA, completely preserving the large genomic fragments required for forensic STR testing, while simultaneously separating most protein and pigment impurities.

[0026] Preferably, the succinic acid compound buffer component is selected from at least two of succinic acid, monosodium succinate, and disodium succinate. The succinic acid binary buffer component has a large buffering capacity, can continuously neutralize basic amine impurities released from putrefactive samples, firmly locks in the optimal weakly neutral region for DNA adsorption, and prevents the complex from dissociating and leaching; furthermore, succinate ions have good compatibility with high concentrations of quaternary ammonium salts and do not inhibit PCR amplification.

[0027] Most existing reagent kits use only a single PEG wash, resulting in incomplete impurity removal. This invention employs a three-stage rinsing system, effectively improving impurity removal efficiency. Preferably, the rinsing solution comprises at least three components: The first rinsing solution has a pH of 6.3–6.5 and is composed of the following ingredients: 10%–15% PEG8000, 400–600 mmol / L sodium chloride, and 25–30 mmol / L MOPS (3-(N-morpholine)propanesulfonic acid). The first stage contains PEG8000 and high-concentration sodium chloride. The large PEG molecule stabilizes the nucleic acid precipitation and washes away free proteins, humic pigments, excess quaternary ammonium salts, and surfactants from the particle surface. The second rinsing solution has a pH of 6.3–6.5 and is composed of the following ingredients: 10%–15% PEG6000, 400–600 mmol / L sodium chloride, and 25–30 mmol / L MOPS. The second stage uses PEG6000 with a smaller molecular weight, which can penetrate into the precipitate to clean up embedded small molecule peptides, humic acid fragments, and other difficult-to-remove impurities. The two-stage MOPS buffer system stably maintains a mild washing environment of pH 6.3–6.5, preventing the dissolution and loss of trace amounts of DNA throughout the process. The third rinsing solution is a 75%–85% (v / v) aqueous ethanol solution. This third-stage ethanol rinsing solution thoroughly washes away residual PEG and inorganic salts, and dehydrates and degreases the product.

[0028] The three-stage gradient washing process of this invention removes macromolecular organic impurities, small molecule inhibitors, salt and polymer residues, significantly reducing PCR amplification interference while preserving large fragments of genomic DNA, thus significantly improving the DNA purity of forensic samples such as hair, nails, old bones, and traces of multiple contamination.

[0029] Preferably, the elution buffer has a pH of 9.0 and a concentration of 5–15 mmol / L Tris-HCl. Washing the dried DNA precipitate disrupts the quaternary ammonium salt-nucleic acid hydrophobic complex, releasing the genomic DNA adsorbed on the vector into the aqueous phase, yielding a pure nucleic acid aqueous solution that can be directly used for PCR amplification; simultaneously, it provides long-term stable storage of the extracted DNA template.

[0030] This invention also provides an extraction method for a forensic genomic DNA extraction reagent system adapted to multiple working conditions, comprising: Mix the sample to be tested, lysis buffer and proteinase K thoroughly, perform lysis treatment, centrifuge after lysis, and collect the supernatant. Add the binding solution to the supernatant, mix well, then add the solid support, and shake to allow the DNA to bind and accumulate on the surface of the solid support, thus obtaining a DNA-loaded solid support. The solid support was fractionated, rinsed, and eluted to obtain the extract.

[0031] The method of this invention is adaptable to various challenging samples under different working conditions: hair, nails, old bones, decaying tissue, and dust / rust / grease-embedded contact traces. It does not require changing the operation steps or adjusting the reagent ratio. The standardized operation is suitable for large-scale forensic evidence testing. The entire operation is simple, with only four core steps: lysis centrifugation, binding enrichment, fractionation rinsing, and elution. It does not require complex filtration or multiple tube transfers, reducing the loss of nucleic acid during the transfer process. It is especially suitable for trace samples.

[0032] Proteinase K can specifically cleave keratin polypeptide chains, breaking down dense, insoluble keratin into soluble small polypeptide molecules, completely disintegrating the keratin matrix, and fully releasing trace amounts of human DNA encapsulated within into the liquid phase, thus significantly improving nucleic acid yield. Therefore, this invention has studied the amount of proteinase K used. Preferably, the volume ratio of the lysis buffer, proteinase K, and binding buffer is 50:1 to 2:30-50 to improve DNA yield.

[0033] The lysis temperature affects enzyme activity and DNA degradation. Therefore, this invention has studied the lysis temperature. Preferably, the lysis conditions are: isothermal lysis at 65-70℃ for 20-25 minutes to improve the lysis effect.

[0034] More specifically, it includes the following steps: (1) Lysis treatment: Add 500 μL of lysis buffer to the sample to be tested, add 10-20 μL of proteinase K, and lyse at 65℃ for 25 min; after lysis, centrifuge and aspirate the supernatant; (2) Binding and enrichment: Add 60% to 100% of the volume of lysis buffer to the supernatant, mix well, add magnetic beads or silica beads solid support, and shake to allow DNA to bind and enrich on the surface of the solid support. (3) Graded rinsing: Select the corresponding rinsing method according to the operating equipment and process, and use the first rinsing solution, the second rinsing solution and the third rinsing solution in sequence to remove impurities; (4) Elution: Use elution buffer to elute DNA from the solid support to obtain the extracted product.

[0035] This method is applicable to DNA extraction from contact trace samples and embedded samples that are contaminated or embedded with dust, pigments, grease, rust, or other contaminants.

[0036] The present invention will be further described in detail below with reference to specific embodiments. It should be noted that the specific embodiments are explanations of the present invention and not limitations thereof.

[0037] Example 1 A forensic genomic DNA extraction reagent system adaptable to multiple working conditions includes a lysis buffer, a binding buffer, a rinsing buffer, and an elution buffer.

[0038] The lysis buffer was a weakly alkaline system containing 30 mmol / L Tris-HCl, 75 mmol / L tetraalkyl quaternary ammonium salt complex (CTAB and DDAC in a molar ratio of 1:1), 22.5 mmol / L lauramide monoethanolamine, 2% Triton X-100 (v / v), and 10 mmol / L EDTA, with the pH value controlled between 7.4 and 7.8.

[0039] The binding solution is a weakly alkaline system containing a 0.75 mol / L tetraalkyl quaternary ammonium salt complex (CTAB and DDAC in a molar ratio of 1:1), 10% PEG8000 (mass fraction), 11.5% alcohol (volume fraction), and a 35 mmol / L succinic acid buffer (succinic acid: disodium succinate in a 1:16 ratio). The initial pH of the binding solution is 7.0–7.1, and after the addition of alcohol, the pH stabilizes at 7.2–7.3.

[0040] In this embodiment, the rinsing solution uses three solutions in the order of use: The first rinsing solution has a pH of 6.3–6.5 and contains 12.5% ​​PEG8000, 500 mmol / L sodium chloride, and 27.5 mmol / L MOPS. The second rinsing solution has a pH of 6.3–6.5 and contains 12.5% ​​PEG6000, 500 mmol / L sodium chloride, and 27.5 mmol / L MOPS. The third rinsing solution is an 80% (v / v) ethanol aqueous solution, used to remove various impurities in stages. The eluent pH was set to 9.0, and the component contained 10 mmol / L Tris-HCl.

[0041] The extraction process is as follows: 1. Lysis treatment: Add 500 μL of lysis buffer and 15 μL of proteinase K to the sample to be tested (10 mg of nail clippings), and lyse at 65℃ for 25 min; after lysis, centrifuge and collect the supernatant. 2. Binding and enrichment: Add 80% of the volume of the lysis buffer to the supernatant, mix well, add magnetic beads, and shake to allow DNA to bind and enrich on the surface of the solid support. 3. Staged rinsing: Impurities are removed by sequentially using the first rinsing solution, the second rinsing solution, and the third rinsing solution; 4. Elution: Elute the DNA from the solid support using an elution buffer to obtain the extracted product.

[0042] Example 2 The sample was blood-stained gauze. The blood-stained area was divided into 2-3 independent segments. A bloodstain carrier the size of a soybean was cut from each segment, avoiding the bloodless blank area. At least one segment was reserved as a backup sample to avoid the complete loss of the sample due to the failure of a single cut.

[0043] This experiment included two control groups with identical rinsing and elution buffers and operating procedures. The lysis buffer and binding buffer were mixed at a 1:1 volume ratio, and the pH of both mixtures was maintained within the range of 6.05–6.15 to eliminate the interference of pH differences on the experimental results. In both systems, the lysis and binding buffers contained identical salts and excipients, except for the characteristic components affecting the buffer system. Specifically: Figure 1 , Figure 3 For the experimental group, the self-releasing acid-base buffer system of the present invention was used. The lysis solution contained laurylamide surfactants, and the binding solution was constructed using a succinic acid compound buffer system. Figure 2 , Figure 4 As a control group, a traditional buffer system was used. The lysis buffer removed the lauramide surfactant, and the binding buffer replaced the succinic acid compound buffer system with a citric acid buffer system.

[0044] The test results show that when the binding solution is present alone, succinic acid and disodium succinate form an acid-base balance and lock the pH at a stable 7.1. When the binding solution is mixed with the alkaline lysis buffer, the alkaline groups in the lysis buffer consume the free hydrogen ions in the system, which drives the succinic acid complex buffer system to continuously dissociate and release acid slowly. The pH of the system then steadily drops back to the exclusive optimal adsorption range for magnetic beads to capture DNA.

[0045] On the other hand, laurylamide amphoteric surfactants can stabilize the distribution of free hydrogen ions in the system, and work synergistically with the succinic acid-based self-release buffer system to precisely constrain the proton release rate, smoothly anchor the pH drop trajectory of the system, and ensure a mild and steady state throughout the self-release acid-base regulation process, avoiding interference from drastic acid-base fluctuations with the specific solid-phase enrichment of DNA. While the specific embodiments of the present invention have been described above, they are not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art without creative effort based on the technical solutions of the present invention are still within the scope of protection of the present invention.

Claims

1. A reagent system for extracting genomic DNA from complex contaminated biological samples adaptable to various working conditions, characterized in that, include: lysis buffer, binding buffer, rinsing buffer, and elution buffer; The lysis buffer is composed of the following raw materials: 20-40 mmol / L Tris-HCl, 50-100 mmol / L tetraalkyl quaternary ammonium salt complex system, 15-30 mmol / L lauramide surfactant, 2%-3% Triton X-100 (volume fraction), 10-15 mmol / L EDTA, and pH 7.4-7.

8.

2. The genomic DNA extraction reagent system for complex contaminated biological samples adapted to multiple operating conditions as described in claim 1, characterized in that, The lauramide surfactant is lauramide monoethanolamine.

3. The genomic DNA extraction reagent system for complex contaminated biological samples adapted to multiple operating conditions as described in claim 1, characterized in that, The binding solution is composed of the following raw materials: a 0.5-1.0 mol / L tetraalkyl quaternary ammonium salt complex system, 10%-15% PEG8000 by mass, 8%-15% alcohol by volume, and 20-50 mmol / L succinic acid complex buffer component, with a pH value of 7.2-7.

3.

4. The genomic DNA extraction reagent system for complex contaminated biological samples adapted to multiple operating conditions as described in claim 3, characterized in that, The alcohol is ethanol or isopropanol.

5. The genomic DNA extraction reagent system for complex contaminated biological samples adapted to multiple operating conditions as described in claim 3, characterized in that, The succinic acid compound buffer component is selected from at least two of succinic acid, monosodium succinate, and disodium succinate.

6. The genomic DNA extraction reagent system for complex contaminated biological samples adapted to multiple operating conditions as described in claim 1, characterized in that, The rinsing solution comprises at least three types: The first rinsing solution has a pH of 6.3–6.5 and is composed of the following ingredients: 10%–15% PEG8000, 400–600 mmol / L sodium chloride, and 25–30 mmol / L MOPS. The second rinsing solution has a pH of 6.3–6.5 and is composed of the following ingredients: 10%–15% PEG6000, 400–600 mmol / L sodium chloride, and 25–30 mmol / L MOPS. The third type of rinsing solution is an aqueous solution of ethanol with a volume fraction of 75%–85%.

7. The genomic DNA extraction reagent system for complex contaminated biological samples adapted to multiple operating conditions as described in claim 1, characterized in that, The eluent has a pH of 9.0 and a concentration of 5–15 mmol / L Tris-HCl.

8. An extraction method for a forensic genomic DNA extraction reagent system adaptable to multiple working conditions, characterized in that, include: Mix the sample to be tested, lysis buffer and proteinase K thoroughly, perform lysis treatment, centrifuge after lysis, and collect the supernatant. Add the binding solution to the supernatant, mix well, then add the solid support, and shake to allow the DNA to bind and accumulate on the surface of the solid support, thus obtaining a DNA-loaded solid support. The solid support was fractionated, rinsed, and eluted to obtain the extract.

9. The extraction method of the forensic genomic DNA extraction reagent system adapted to multiple working conditions as described in claim 8, characterized in that, The volume ratio of the lysis buffer, proteinase K, and binding buffer is 50:1 to 2:30-50. Alternatively, the pyrolysis conditions are: isothermal pyrolysis at 65-70℃ for 20-25 minutes.

10. The application of the reagent system according to any one of claims 1-7 in forensic genomic DNA extraction, characterized in that, The samples include: At least one of the following: old bones, decaying tissue, hair, and nails.

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