Decalcification-free trace DNA extraction method for old bone detection material

Abstract

According to the decalcification-free trace DNA extraction method for the old bone sample, an independent decalcification step is not adopted, a specific metal chelating agent and a phosphoramidate derivative are introduced into a bone incubation solution, the specific metal chelating agent has high chelating capacity on calcium and magnesium ions, and the specific metal chelating agent and a complex product thereof are high in water solubility and not prone to being separated out; the phosphoramidate derivative is easy to remove and does not affect the DNA adsorption efficiency of the magnetic beads and subsequent PCR amplification, the phosphoramidate derivative preferentially acts with a phosphate group fixed site on the surface of the soil, adsorption of the soil to the DNA is greatly reduced, an incubation product can be obtained to the maximum extent, then soil impurities are removed through centrifugation, and it is ensured that no adverse effect is generated on subsequent DNA adsorption of the magnetic beads. According to the method, trace DNA can be effectively extracted and purified from old bone detection materials, independent decalcification pretreatment is not needed, sample loss is avoided, and the extracted DNA can be used for forensic medicine identification.

Description

Technical Field

[0001] The present invention relates to the field of biological and medical testing technology, more specifically, to the field of DNA extraction technology for old bone samples, and in particular to a method for extracting trace amounts of DNA from old bone samples without decalcification. Background Art

[0002] Bones are a special type of biological specimen. They are a dense connective tissue that contains a large amount of calcium compounds, which greatly slows down the decomposition of cells or DNA inside the bones by microorganisms, making bone specimens decay and degrade more slowly than other tissues and organs in the human body. Therefore, in actual forensic cases, bones are the only biological specimens for DNA testing of highly decayed and skeletal corpses.

[0003] Most of the old skeletal samples are those that have been buried for a long time. They have been preserved for a long time and in a complex environment. As a result, the old skeletal samples have problems such as severe sample degradation, high content of soil impurities, and high content of metal impurities, making old skeletal samples a difficulty in the actual forensic case inspection.

[0004] For old bone samples, traditional methods often require increasing the amount of bone powder to increase the total amount of sample DNA, but this also introduces too many impurities. In order to eliminate the influence of calcium in bone powder and metal impurities in soil, it is necessary to use 0.5mol / L EDTA decalcification solution (pH 8.0) to decalcify for 12 hours before incubating the bone powder, then centrifuge and discard the decalcification solution before incubating the bone powder. If the EDTA decalcification solution is directly introduced into the bone incubation solution, the product will be unstable after the excessive metal impurities are complexed with the decalcification solution, and it is easy to precipitate fine particle impurities under the extraction environment, which will affect the efficiency of magnetic beads to adsorb DNA. If the metal ions or precipitated impurities cannot be effectively removed, it will affect the subsequent PCR amplification. Therefore, the traditional method uses a separate decalcification method, but it will cause the DNA in the bone cells on the surface of the bone chips to be lost during the decalcification process, affecting the detection effect of old bone samples and increasing the extraction test time.

[0005] Therefore, in view of the above situation, it is necessary to develop a method for extracting trace DNA from old bone samples without decalcification, which can effectively extract and purify trace DNA from old bone samples without the need for separate decalcification pre-treatment to avoid sample loss. The extracted DNA can be used for forensic identification and can provide a more effective basis for case detection and court trials. Summary of the invention

[0006] In order to overcome the shortcomings of the above-mentioned prior art, one purpose of the present invention is to provide a method for extracting trace DNA from old bone samples without decalcification, which can effectively extract and purify trace DNA from old bone samples without the need for separate decalcification pre-treatment, thereby avoiding sample loss. The extracted DNA can be used for forensic identification, which can provide a more effective basis for case detection and court trials, and is suitable for large-scale promotion and application.

[0007] Another object of the present invention is to provide a method for extracting trace amounts of DNA from old bone samples without decalcification. The method has an ingenious design, a unique concept, and is easy to operate. It eliminates the need for a separate decalcification step, simplifies the operating process, shortens the extraction time, and is suitable for large-scale promotion and application.

[0008] To achieve the above purpose, the present invention provides a method for extracting trace DNA from old bone samples without decalcification, which is characterized by comprising the following steps:

[0009] (1) Incubation: Mix bone powder, bone incubation solution, proteinase K solution and DTT solution obtained from old bone samples and incubate them, shake them vigorously several times during the process, and centrifuge to obtain a first supernatant, wherein the bone incubation solution comprises 0.05wt% to 0.2wt% of sodium dodecyl sulfate, 5mmol / L to 15mmol / L of EDTA, 10mmol / L to 40mmol / L of Tris-HCl, 0.1mol / L to 0.5mol / L of a specific metal chelator and 0.05mmol / L to 0.3mmol / L of an aminophosphoric acid ester derivative, the pH value of the bone incubation solution is 7.5 to 8.5, and the specific metal chelator is selected from one or more of tetrasodium iminodisuccinate, tetrasodium glutamate diacetate, trisodium N,N-dicarboxymethylalanine, sodium gluconate and sodium ethylenediamine di-o-phenylacetate;

[0010] (2) adding a carboxylic acid-based cationic resin to the first supernatant, mixing well, placing in a metal bath, and centrifuging to obtain a second supernatant;

[0011] (3) adding bone adsorption liquid and magnetic bead suspension to the second supernatant, turning over and mixing for adsorption, and then magnetically aspirating to discard all liquid to obtain magnetic beads adsorbed with DNA;

[0012] (4) Washing and eluting the magnetic beads adsorbed with DNA to obtain a DNA solution of the old bone sample.

[0013] Preferably, in step (1), the bone powder is 1g to 3g, the bone incubation solution is 3mL to 5mL, the proteinase K solution is 100uL to 300uL, the concentration of the proteinase K solution is 20mg / mL, the DTT solution is 20uL to 60uL, the concentration of the DTT solution is 1mol / L, the incubation temperature is 37°C to 56°C, the incubation time is 1h to 12h, the number of vigorous shaking is 5 to 8 times, the centrifugal speed is 7000rpm to 10000rpm, and the centrifugal time is 5 minutes to 8 minutes.

[0014] Preferably, in step (1), the amino phosphoroester derivative is selected from one or more of α-amino phosphoroester, β-amino phosphoroester and γ-amino phosphoroester, wherein the amino group is an unsubstituted amino group, a monosubstituted amino group or a disubstituted amino group, the substituent of the amino group is an aliphatic group, and the amino phosphoroester is a fatty alcohol phosphate with a length of less than 5 carbon atoms.

[0015] Preferably, in step (2), the carboxylic acid-based cationic resin is 10 mg to 50 mg, the temperature of the metal bath is 60° C. to 90° C., the time of the metal bath is 10 minutes to 20 minutes, the speed of the centrifuge is 7000 rpm to 10000 rpm, and the time of the centrifuge is 2 minutes to 4 minutes.

[0016] Preferably, in step (3), the bone adsorption solution is 8 mL to 15 mL, the bone adsorption solution includes 3.0 mol / L to 6.0 mol / L guanidine isothiocyanate, 10 mmol / L to 40 mmol / L Tris-HCl and 1.5 mol / L to 3 mol / L ethylene glycol, the pH value of the bone adsorption solution is 3 to 5, the magnetic bead suspension is 30 uL to 60 uL, the flipping mixing adsorption time is 20 minutes to 30 minutes, and the magnetic absorption time is 5 minutes to 10 minutes;

[0017] Preferably, in step (3), the magnetic bead suspension includes 9 wt% of silica magnetic composite particles with a size of 100 nm to 2000 nm, wherein the silica magnetic composite particles are cross-linked bodies formed by cross-linking two or more ferrite-silica core-shell nanoparticles, and the surface of the silica magnetic composite particles has a plurality of nanoscale protrusions.

[0018] Preferably, in step (4), the specific steps of washing include: using washing solution I to perform a first washing and magnetic absorption on the magnetic beads adsorbed with DNA, and discarding all liquid; using washing solution II to perform a second washing and magnetic absorption, and discarding all liquid; using washing solution III to perform a third washing and magnetic absorption, and discarding all liquid.

[0019] More preferably, in step (4), the washing solution I comprises 2mol / L to 5mol / L of guanidine thiocyanate, 5mmol / L to 25mmol / L of Tris-HCl and 10mmol / L to 50mmol / L of a specific metal chelator, and the amount of the washing solution I used is 300uL to 700uL. The washing solution II comprises 2mol / L of sodium chloride, 50% by volume of anhydrous ethanol, 20% by volume of isopropanol and 30% by volume of water, and the amount of the washing solution II used is 600uL to 1000uL. The washing solution III comprises 50% by volume of anhydrous ethanol and 50% by volume of isopropanol, and the amount of the washing solution III used is 600uL to 1000uL.

[0020] Preferably, in step (4), the specific step of eluting includes: drying the magnetic beads adsorbed with DNA and then adding water to mix and dissolve them, and the liquid obtained after mixing and magnetic absorption is the DNA solution.

[0021] More preferably, in step (4), the drying is performed in a 65°C metal bath for 5 to 10 minutes, the amount of water used is 20uL to 100uL, the dissolution is performed in a 65°C metal bath for 10 minutes, and the magnetic attraction time is 2 minutes.

[0022] The beneficial effects of the present invention are:

[0023] 1. The decalcification-free trace DNA extraction method for old bone specimens of the present invention does not adopt a separate decalcification step, and introduces specific metal chelators and aminophosphoric acid ester derivatives into the bone incubation solution. The specific metal chelators have strong chelating ability for calcium and magnesium ions, and the specific metal chelators and their complex products are highly water-soluble, not easy to precipitate, and easy to remove, and will not affect the efficiency of magnetic bead adsorption of DNA and subsequent PCR amplification. The aminophosphoric acid ester derivatives preferentially act on the fixed sites of phosphate groups on the soil surface, greatly reducing the adsorption of DNA by the soil, and can obtain incubation products to the maximum extent. Subsequently, the soil impurities are removed by centrifugation to ensure that there is no adverse effect on the subsequent magnetic bead adsorption of DNA. Therefore, it can effectively extract and purify trace DNA from old bone specimens without the need for separate decalcification pretreatment, avoiding sample loss, and the extracted DNA can be used for forensic identification, which can provide a more effective basis for case detection and court trials, and is suitable for large-scale promotion and application.

[0024] 2. The decalcification-free trace DNA extraction method for old bone specimens of the present invention does not adopt a separate decalcification step, and introduces specific metal chelators and aminophosphoric acid ester derivatives into the bone incubation solution. The specific metal chelators have a strong chelating ability for calcium and magnesium ions, and the specific metal chelators and their complex products are highly water-soluble, not easy to precipitate, and easy to remove, and will not affect the efficiency of magnetic bead adsorption of DNA and subsequent PCR amplification. The aminophosphoric acid ester derivatives preferentially act on the fixed sites of phosphate groups on the soil surface, greatly reducing the adsorption of DNA by the soil, and can obtain the incubation product to the maximum extent. Subsequently, the soil impurities are removed by centrifugation to ensure that there is no adverse effect on the subsequent magnetic bead adsorption of DNA. Therefore, the design is ingenious, the conception is unique, the operation is simple, the separate decalcification step is eliminated, the operation process is simplified, the extraction time is shortened, and it is suitable for large-scale promotion and application.

[0025] These and other objects, features and advantages of the present invention will be more fully reflected in the following detailed description and claims, and may be achieved by the means, devices and combinations thereof particularly pointed out in the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the STR profile of the DNA from the old bone sample extracted in Example 1.

[0027] Figure 2 This is the STR profile of the DNA from the old bone sample extracted in Example 2.

[0028] Figure 3 This is the STR profile of the DNA from the old bone sample extracted in Example 3.

[0029] Figure 4 This is the STR spectrum of the DNA extracted from the old bone sample in Example 1.

[0030] Figure 5 This is the STR spectrum of the DNA extracted from the old bone sample in Example 2.

[0031] Figure 6 This is the STR spectrum of the DNA extracted from the old bone sample in Example 3. DETAILED DESCRIPTION

[0032] In order to simplify the operational process of DNA extraction from old bone samples, eliminate the separate decalcification step, shorten the extraction time, and at the same time ensure that the extracted DNA can meet the requirements of forensic identification and provide an effective basis for case detection and court trials, the inventors have invented a method for extracting trace DNA from old bone samples without decalcification through in-depth and extensive research. The method for extracting trace DNA from old bone samples without decalcification is ingeniously designed and uniquely conceived. It can effectively extract and purify trace DNA from old bone samples, does not require separate decalcification pre-treatment, avoids sample loss, simplifies the operational process, shortens the extraction time, and the extracted DNA can be used for forensic identification, which can provide a more effective basis for case detection and court trials.

[0033] The method for extracting trace DNA from old bone samples without decalcification of the present invention comprises the following steps:

[0034] (1) Incubation: Mix bone powder, bone incubation solution, proteinase K solution and DTT solution obtained from old bone samples and incubate them, shake them vigorously several times during the process, and centrifuge to obtain a first supernatant, wherein the bone incubation solution comprises 0.05wt% to 0.2wt% of sodium dodecyl sulfate, 5mmol / L to 15mmol / L of EDTA, 10mmol / L to 40mmol / L of Tris-HCl, 0.1mol / L to 0.5mol / L of a specific metal chelator and 0.05mmol / L to 0.3mmol / L of an aminophosphoric acid ester derivative, the pH value of the bone incubation solution is 7.5 to 8.5, and the specific metal chelator is selected from one or more of tetrasodium iminodisuccinate, tetrasodium glutamate diacetate, trisodium N,N-dicarboxymethylalanine, sodium gluconate and sodium ethylenediamine di-o-phenylacetate;

[0035] (2) adding a carboxylic acid-based cationic resin to the first supernatant, mixing well, placing in a metal bath, and centrifuging to obtain a second supernatant;

[0036] (3) adding bone adsorption liquid and magnetic bead suspension to the second supernatant, turning over and mixing for adsorption, and then magnetically aspirating to discard all liquid to obtain magnetic beads adsorbed with DNA;

[0037] (4) Washing and eluting the magnetic beads adsorbed with DNA to obtain a DNA solution of the old bone sample.

[0038] In the step (1), the amount of the bone powder, the amount of the bone incubation solution, the amount and concentration of the proteinase K solution, the amount and concentration of the DTT solution, the incubation temperature and time, the number of times of vigorous shaking, and the speed and time of the centrifugation can be determined as needed. Preferably, in the step (1), the bone powder is 1g to 3g, the bone incubation solution is 3mL to 5mL, the proteinase K solution is 100uL to 300uL, the concentration of the proteinase K solution is 20mg / mL, the DTT solution is 20uL to 60uL, the concentration of the DTT solution is 1mol / L, the incubation temperature is 37°C to 56°C, the incubation time is 1h to 12h, the number of times of vigorous shaking is 5 to 8 times, the speed of the centrifugation is 7000rpm to 10000rpm, and the centrifugation time is 5 minutes to 8 minutes.

[0039] In the step (1), the amino phosphoroester derivative can be any suitable specific amino phosphoroester derivative. Preferably, in the step (1), the amino phosphoroester derivative is selected from one or more of α-amino phosphoroester, β-amino phosphoroester and γ-amino phosphoroester, wherein the amino group is an unsubstituted amino group, a monosubstituted amino group or a disubstituted amino group, the substituent of the amino group is an aliphatic group, and the amino phosphoroester is a fatty alcohol phosphate with a length of less than 5 carbon atoms.

[0040] In the step (2), the amount of the carboxylic acid-based cationic resin, the temperature and time of the metal bath, and the speed and time of the centrifugation can be determined as needed. Preferably, in the step (2), the carboxylic acid-based cationic resin is 10 mg to 50 mg, the temperature of the metal bath is 60° C. to 90° C., the time of the metal bath is 10 minutes to 20 minutes, the speed of the centrifugation is 7000 rpm to 10000 rpm, and the time of the centrifugation is 2 minutes to 4 minutes.

[0041] In the step (3), the amount and composition of the bone adsorption solution, the amount of the magnetic bead suspension, the time of the inversion mixing adsorption and the time of the magnetic attraction can be determined as needed. Preferably, in the step (3), the bone adsorption solution is 8mL to 15mL, the bone adsorption solution includes 3.0mol / L to 6.0mol / L guanidine isothiocyanate, 10mmol / L to 40mmol / L Tris-HCl and 1.5mol / L to 3mol / L ethylene glycol, the pH value of the bone adsorption solution is 3 to 5, the magnetic bead suspension is 30uL to 60uL, the inversion mixing adsorption time is 20 minutes to 30 minutes, and the magnetic attraction time is 5 minutes to 10 minutes;

[0042] In the step (3), the magnetic bead suspension can be any suitable magnetic beads. Preferably, in the step (3), the magnetic bead suspension includes 9wt% of silica magnetic composite particles with a size of 100nm to 2000nm, the silica magnetic composite particles are cross-linked bodies formed by cross-linking two or more ferrite-silica core-shell nanoparticles, and the surface of the silica magnetic composite particles has a plurality of nano-scale protrusions. The synthesis method of the silica magnetic composite particles can be specifically referred to Chinese invention patent application CN200810050941.0.

[0043] In the step (3), the silica magnetic composite particles may be any suitable silica magnetic composite particles. More preferably, in the step (3), the silica magnetic composite particles are Fe3O4-silica magnetic composite particles.

[0044] In the step (4), the washing may be performed by any suitable specific steps. Preferably, in the step (4), the specific steps of the washing include: using washing solution I to perform a first washing and magnetic absorption on the magnetic beads adsorbed with DNA, and discarding all liquid; using washing solution II to perform a second washing and magnetic absorption, and discarding all liquid; using washing solution III to perform a third washing and magnetic absorption, and discarding all liquid.

[0045] In the step (4), the composition and amount of the washing solution I, the composition and amount of the washing solution II, and the composition and amount of the washing solution III can be determined as needed. More preferably, in the step (4), the washing solution I includes 2mol / L to 5mol / L of guanidine thiocyanate, 5mmol / L to 25mmol / L of Tris-HCl and 10mmol / L to 50mmol / L of a specific metal chelator, the amount of the washing solution I is 300uL to 700uL, the washing solution II includes 2mol / L of sodium chloride, 50% by volume of anhydrous ethanol, 20% by volume of isopropanol and 30% by volume of water, the amount of the washing solution II is 600uL to 1000uL, the washing solution III includes 50% by volume of anhydrous ethanol and 50% by volume of isopropanol, and the amount of the washing solution III is 600uL to 1000uL.

[0046] In the step (4), the elution may be carried out by any suitable specific steps. Preferably, in the step (4), the specific steps of the elution include: drying the magnetic beads adsorbed with DNA and then adding water to mix and dissolve them. The liquid obtained after mixing and magnetic absorption is the DNA solution.

[0047] In the step (4), the drying and the dissolving may adopt any suitable conditions, and the amount of water used and the time of the magnetic attraction may be determined as needed. More preferably, in the step (4), the drying adopts a 65°C metal bath for 5 minutes to 10 minutes, the amount of water used is 20uL to 100uL, the dissolving adopts a 65°C metal bath for 10 minutes, and the time of the magnetic attraction is 2 minutes.

[0048] In order to more clearly understand the technical content of the present invention, the following measurement examples are given to explain in detail.

[0049] The experimental methods in the following examples without specifying specific conditions are usually measured according to national standards. If there are no corresponding national standards, they are carried out according to the general international standards, conventional conditions, or conditions recommended by the manufacturer.

[0050] Unless otherwise defined or indicated, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the method of the present invention.

[0051] Example 1

[0052] 1. Reagent materials:

[0053] 1) a bone incubation solution comprising 0.05wt% sodium dodecyl sulfate, 15mmol / L EDTA, 10mmol / L Tris-HCl, 0.5mol / L specific metal chelator and 0.05mmol / L aminophosphoric acid ester derivative, the pH value of which is adjusted to 8.0 with 1mol / L NaOH solution, the specific metal chelator is tetrasodium glutamate diacetate, and the aminophosphoric acid ester derivative is α-aminophosphoric acid ester;

[0054] 2) The bone adsorption solution includes 6.0 mol / L guanidine thiocyanate, 10 mmol / L Tris-HCl and 3 mol / L ethylene glycol, and the pH value is adjusted to 4.0 with acetic acid-sodium acetate buffer solution;

[0055] 3) The magnetic bead suspension contains 9% by mass of Fe3O4-silicon dioxide magnetic composite particles with a size of 600 nm dispersed in high-purity water. The Fe3O4-silicon dioxide magnetic composite particles are prepared by the method disclosed in Chinese invention patent application CN200810050941.0, and the specific steps are as follows:

[0056] Utilize the method disclosed in the literature (Albert P. Philipse, Magnetic Silica Dispersons: Preparation and Stability of Surface-Modified Silica Particles with a Magnetic Core, Langmuir 1994, 10, 92-99) to synthesize 30nm Fe3O4-silicon dioxide core-shell nanoparticles. Take 500ml water and 500ml propanol and mix them, then add 50.0g aluminum chloride and 2.0g tetraethylene pentamine, stir and dissolve, then add 5.0g 30nm Fe3O4-silicon dioxide core-shell nanoparticles, and react for 15 minutes under the condition of stirring speed of 50rpm. Then add 30ml of concentrated ammonia water with a mass percentage concentration of 25-28% in the solution, and under the stirring speed of 300rpm, add 0.2ml of tetraethyl orthosilicate dropwise, and react for 12 hours. The solid was separated from the reaction solution by a permanent magnet and washed three times with high-purity water to obtain Fe3O4-silicon dioxide magnetic composite particles with an average size of 600 nm.

[0057] 4) Washing solution I includes 5 mol / L guanidine thiocyanate, 10 mmol / L Tris-HCl and 50 mmol / L specific metal chelator, with a pH value of 7.0. The specific metal chelator is tetrasodium glutamate diacetate.

[0058] 5) washing liquid II comprises 2 mol / L sodium chloride, 50% by volume of anhydrous ethanol, 20% by volume of isopropanol and 30% by volume of water;

[0059] 6) the washing liquid III comprises 50% by volume of anhydrous ethanol and 50% by volume of isopropanol;

[0060] 2. Experimental steps

[0061] 1) Take a piece of old bone sample that has been buried for more than 30 years, clean it, and prepare it into bone powder for later use;

[0062] 2) Take a screw-capped centrifuge tube and weigh 3g of bone powder, add 5mL of bone incubation solution, 200uL of proteinase K solution (20mg / mL), and 50uL of DTT solution (1mol / L), shake and mix, and place in a water bath at 51°C for 6 hours, shaking vigorously 5 times. Then put the centrifuge tube into a centrifuge at 7000rpm for 5 minutes, and transfer the supernatant (i.e., the first supernatant) to another centrifuge tube;

[0063] 3) Add 50 mg of carboxylic acid-based cationic resin to the supernatant, shake and mix, place in a metal bath at 80° C. for 20 minutes, and then centrifuge at 7000 rpm for 3 minutes. Pipet the supernatant (i.e., the second supernatant) and transfer to another centrifuge tube;

[0064] 4) Add 10 mL of bone adsorption solution to the supernatant, then add 50 uL of magnetic bead suspension, shake and mix, place the centrifuge tube on an automatic flip mixer, flip and mix to adsorb for 30 minutes, immediately put it back on the magnetic stand after adsorption, magnetically absorb for 5 minutes, then discard all the liquid to obtain magnetic beads adsorbed with DNA;

[0065] 5) Add 600uL of washing solution I to the centrifuge tube, shake and mix, immediately put it back on the magnetic stand, magnetically absorb for 2 minutes, and discard all the liquid; then add 800uL of washing solution II, shake and mix, immediately put it back on the magnetic stand, magnetically absorb for 2 minutes, and discard all the liquid; finally, add 800uL of washing solution III, shake and mix, immediately put it back on the magnetic stand, magnetically absorb for 2 minutes, and discard all the liquid;

[0066] 6) Place the centrifuge tube with the lid open in a 65°C metal bath to dry for 5 minutes. After the contents are completely dry, add 20uL of high-purity water to disperse all the magnetic beads into the solution. Close the lid and warm in a 65°C metal bath for 10 minutes. After shaking and mixing, immediately place it back on the magnetic rack and magnetically absorb for 2 minutes. Aspirate the DNA solution and save it in another centrifuge tube.

[0067] The total extraction time was approximately 8 hours.

[0068] The DNA solution extracted and purified in Example 1 was subjected to PCR amplification using the FastDirect DNA 25A amplification kit (Baiteyuan Biotechnology Co., Ltd.), and electrophoresis was performed in an ABI 3500XL instrument. The STR typing spectrum obtained is as follows: Figure 1 As shown, the STR loci are complete and accurately typed, among which 25 loci alleles are complete and the peak heights are all above 2000, which meets the requirements of forensic case identity identification detection data. This shows that the purification method has a short extraction time, high purification efficiency, and high purity of the obtained DNA, which is very suitable for the extraction of trace DNA from old bone samples.

[0069] Example 2

[0070] 1. Reagent materials:

[0071] 1) a bone incubation solution comprising 0.1wt% sodium dodecyl sulfate, 10mmol / L EDTA, 40mmol / L Tris-HCl, 0.1mol / L specific metal chelator and 0.3mmol / L aminophosphoric acid ester derivative, the pH value of which is adjusted to 7.5 with 1mol / L NaOH solution, the specific metal chelator is sodium gluconate, and the aminophosphoric acid ester derivative is β-aminophosphoric acid ester;

[0072] 2) the bone adsorption solution includes 5.0 mol / L guanidine thiocyanate, 30 mmol / L Tris-HCl and 2 mol / L ethylene glycol, and the pH value is adjusted to 5.0 with acetic acid-sodium acetate buffer solution;

[0073] 3) The magnetic bead suspension contained 9% by weight of silica carboxyl magnetic beads (Suzhou Beaver Biomedical Engineering Co., Ltd.) with a size of 600 nm dispersed in high-purity water.

[0074] 4) Washing solution I includes 2 mol / L guanidine thiocyanate, 5 mmol / L Tris-HCl and 30 mmol / L specific metal chelator, with a pH value of 7.0, and the specific metal chelator is sodium gluconate.

[0075] 5) washing liquid II comprises 2 mol / L sodium chloride, 50% by volume of anhydrous ethanol, 20% by volume of isopropanol and 30% by volume of water;

[0076] 6) the washing liquid III comprises 50% by volume of anhydrous ethanol and 50% by volume of isopropanol;

[0077] 2. Experimental steps

[0078] 1) The bone powder is the bone powder prepared in Example 1;

[0079] 2) Take a screw-cap centrifuge tube and weigh 1g of bone powder, add 4mL of bone incubation solution, 300uL of proteinase K solution (20mg / mL), and 20uL of DTT solution (1mol / L), shake and mix, and place in a water bath at 37℃ for 12 hours, shaking vigorously 7 times during the period. Then put the centrifuge tube into a centrifuge and centrifuge at 8000rpm for 6 minutes, and transfer the supernatant (i.e., the first supernatant) to another centrifuge tube;

[0080] 3) Add 30 mg of carboxylic acid-based cationic resin to the supernatant, shake and mix, place in a metal bath at 60° C. for 15 minutes, and then centrifuge at 10,000 rpm for 2 minutes. Pipet the supernatant (i.e., the second supernatant) and transfer to another centrifuge tube;

[0081] 4) Add 8 mL of bone adsorption solution to the supernatant, then add 60 uL of magnetic bead suspension, shake and mix, place the centrifuge tube on an automatic flip mixer, flip and mix to adsorb for 25 minutes, immediately put it back on the magnetic stand after adsorption, magnetically absorb for 8 minutes, then discard all the liquid to obtain magnetic beads adsorbed with DNA;

[0082] 5) Add 700uL of washing solution I to the centrifuge tube, shake and mix, immediately put it back on the magnetic stand, magnetically absorb for 2 minutes, and discard all the liquid; then add 600uL of washing solution II, shake and mix, immediately put it back on the magnetic stand, magnetically absorb for 2 minutes, and discard all the liquid; finally, add 1000uL of washing solution III, shake and mix, immediately put it back on the magnetic stand, magnetically absorb for 2 minutes, and discard all the liquid;

[0083] 6) Open the centrifuge tube and place it in a 65℃ metal bath to dry for 7 minutes. After the contents are completely dry, add 100uL of high-purity water to disperse all the magnetic beads into the solution. Close the cover and warm it in a 65℃ metal bath for 10 minutes. After shaking and mixing, immediately put it back on the magnetic rack and magnetically absorb it for 2 minutes. Aspirate the DNA solution and save it in another centrifuge tube.

[0084] The total extraction time was approximately 14 hours.

[0085] The DNA solution extracted and purified in Example 2 was subjected to PCR amplification using the FastDirect DNA 25A amplification kit (Baiteyuan Biotechnology Co., Ltd.), and electrophoresis was performed in an ABI 3500XL instrument. The STR typing spectrum obtained was as shown in FIG. Figure 2 As shown, the STR loci are complete and accurately typed, among which 25 loci alleles are complete and the peak heights are all above 2000, which meets the requirements of forensic case identity identification detection data. This shows that the purification method has a short extraction time, high purification efficiency, and high purity of the obtained DNA, which is very suitable for the extraction of trace DNA from old bone samples.

[0086] Example 3

[0087] 1. Reagent materials:

[0088] 1) a bone incubation solution comprising 0.2wt% sodium dodecyl sulfate, 5mmol / L EDTA, 20mmol / L Tris-HCl, 0.3mol / L specific metal chelator and 0.1mmol / L aminophosphoric acid ester derivative, the pH value of which is adjusted to 8.5 with 1mol / L NaOH solution, the specific metal chelator is sodium ethylenediamine di-o-phenylacetate, and the aminophosphoric acid ester derivative is γ-aminophosphoric acid ester;

[0089] 2) The bone adsorption solution includes 3.0 mol / L guanidine thiocyanate, 40 mmol / L Tris-HCl and 1.5 mol / L ethylene glycol, and the pH value is adjusted to 3.0 with acetic acid-sodium acetate buffer solution;

[0090] 3) The magnetic bead suspension contained 9% by weight of silica carboxyl magnetic beads (Suzhou Beaver Biomedical Engineering Co., Ltd.) with a size of 600 nm dispersed in high-purity water.

[0091] 4) Washing solution I includes 3 mol / L guanidine thiocyanate, 25 mmol / L Tris-HCl and 10 mmol / L specific metal chelating agent, with a pH value of 7.0. The specific metal chelating agent is sodium ethylenediamine di-o-phenylacetate.

[0092] 5) washing liquid II comprises 2 mol / L sodium chloride, 50% by volume of anhydrous ethanol, 20% by volume of isopropanol and 30% by volume of water;

[0093] 6) the washing liquid III comprises 50% by volume of anhydrous ethanol and 50% by volume of isopropanol;

[0094] 2. Experimental steps

[0095] 1) The bone powder is the bone powder prepared in Example 1;

[0096] 2) Take a screw-cap centrifuge tube and weigh 2g of bone powder, add 3mL of bone incubation solution, 100uL of proteinase K solution (20mg / mL), and 60uL of DTT solution (1mol / L), shake and mix, and place in a water bath at 56℃ for 1 hour, shaking vigorously 8 times. Then put the centrifuge tube into a centrifuge at 10000rpm for 8 minutes, and transfer the supernatant (i.e., the first supernatant) to another centrifuge tube;

[0097] 3) Add 10 mg of carboxylic acid-based cationic resin to the supernatant, shake and mix, place in a metal bath at 90° C. for 10 minutes, and then centrifuge at 9000 rpm for 4 minutes. Pipet the supernatant (i.e., the second supernatant) and transfer to another centrifuge tube;

[0098] 4) Add 15 mL of bone adsorption solution to the supernatant, then add 30 uL of magnetic bead suspension, shake and mix, place the centrifuge tube on an automatic flip mixer, flip and mix for 20 minutes, and immediately put it back on the magnetic stand after adsorption, magnetically absorb for 10 minutes, then discard all the liquid to obtain magnetic beads adsorbed with DNA;

[0099] 5) Add 300uL of washing solution I to the centrifuge tube, shake and mix, immediately put it back on the magnetic stand, magnetically absorb for 2 minutes, and discard all the liquid; then add 1000uL of washing solution II, shake and mix, immediately put it back on the magnetic stand, magnetically absorb for 2 minutes, and discard all the liquid; finally, add 600uL of washing solution III, shake and mix, immediately put it back on the magnetic stand, magnetically absorb for 2 minutes, and discard all the liquid;

[0100] 6) Place the centrifuge tube with the lid open in a 65°C metal bath to dry for 10 minutes. After the contents are completely dry, add 50uL of high-purity water to disperse all the magnetic beads into the solution. Close the lid and warm in a 65°C metal bath for 10 minutes. After shaking and mixing, immediately place it back on the magnetic rack and magnetically absorb for 2 minutes. Aspirate the DNA solution and save it in another centrifuge tube.

[0101] The total extraction time was approximately 3 hours.

[0102] The DNA solution extracted and purified in Example 3 was subjected to PCR amplification using the FastDirect DNA 25A amplification kit (Baiteyuan Biotechnology Co., Ltd.), and electrophoresis was performed in an ABI 3500XL instrument. The STR typing spectrum obtained was as shown in the figure. Figure 3 As shown, the STR loci are complete and accurately typed, among which 25 loci alleles are complete, and the peak heights are all above 1500, which meets the requirements of forensic case identity identification detection data. This shows that the purification method has a short extraction time, high purification efficiency, and high purity of the obtained DNA, which is very suitable for the extraction of trace DNA from old bone samples.

[0103] Comparative Example 1

[0104] 1. Reagent materials:

[0105] 1) The bone incubation solution includes 0.05 wt % sodium dodecyl sulfate, 15 mmol / L EDTA and 10 mmol / L Tris-HCl, and the pH value is adjusted to 8.0 with 1 mol / L NaOH solution.

[0106] 2) The bone adsorption solution includes 6.0 mol / L guanidine thiocyanate, 10 mmol / L Tris-HCl and 3 mol / L ethylene glycol, and the pH value is adjusted to 4.0 with acetic acid-sodium acetate buffer solution.

[0107] 3) The magnetic bead suspension contained 9% by weight of silica carboxyl magnetic beads (Suzhou Beaver Biomedical Engineering Co., Ltd.) with a size of 600 nm dispersed in high-purity water.

[0108] 4) The washing liquid I comprises 80% by volume of anhydrous ethanol and 20% by volume of water.

[0109] 5) The washing liquid II includes 80% by volume of anhydrous ethanol and 20% by volume of water.

[0110] 6) The washing liquid III includes 80% by volume of anhydrous ethanol and 20% by volume of water.

[0111] Comparison of the reagent materials used in Comparative Example 1 and Example 1 of the present invention:

[0112]

[0113] 2. Experimental steps

[0114] 1) The bone powder was prepared in Example 1.

[0115] 2) Take 3g of bone powder in a screw-cap centrifuge tube, add 5mL of bone incubation solution, 200uL of proteinase K (20mg / mL), and 50uL of DTT (1mol / L), shake and mix, and place in a water bath at 51°C for 6 hours, shaking vigorously 5 times. Then place the centrifuge tube in a centrifuge at 7000rpm for 5 minutes, and transfer the supernatant to another centrifuge tube.

[0116] 3) Add 10 mL of bone adsorption solution to the supernatant, then add 50 uL of magnetic bead suspension, shake and mix, place the centrifuge tube on an automatic flip mixer, flip and mix to adsorb for 30 minutes, put it back on the magnetic stand immediately after adsorption, magnetically adsorb for 5 minutes, then discard all the liquid to obtain magnetic beads adsorbed with DNA.

[0117] 4) Add 600uL of washing solution I to the centrifuge tube, shake and mix, immediately put it back on the magnetic stand, magnetically absorb for 2 minutes, and discard all the liquid; then add 800uL of washing solution II, shake and mix, immediately put it back on the magnetic stand, magnetically absorb for 2 minutes, and discard all the liquid; finally, add 800uL of washing solution III, shake and mix, immediately put it back on the magnetic stand, magnetically absorb for 2 minutes, and discard all the liquid;

[0118] 5) Open the centrifuge tube and place it in a 65℃ metal bath to dry for 5 minutes. Then add 20uL of high-purity water to disperse all the magnetic beads into the solution. Close the cover and warm it in a 65℃ metal bath for 10 minutes. After shaking and mixing, immediately put it back on the magnetic rack and magnetically absorb it for 2 minutes. Aspirate the DNA solution and save it in another centrifuge tube.

[0119] The total extraction time was approximately 7.5 hours.

[0120] The DNA solution obtained in Comparative Example 1 was amplified by PCR using the FastDirect DNA 25A Amplification Kit (Baiteyuan Biotechnology Co., Ltd.), and electrophoresed in an ABI 3500XL instrument. The STR typing spectrum obtained is as shown in FIG. Figure 4As shown, the STR loci are incomplete and none of the 25 gene loci were detected, indicating that the purification method has a short extraction time and low purification efficiency, and cannot obtain DNA with purity that meets the requirements. The integrity is poor and it is not suitable for extracting trace DNA from old bone samples.

[0121] Comparative Example 2

[0122] 1. Reagent materials:

[0123] 1) The bone incubation solution includes 0.05 wt% sodium dodecyl sulfate, 15 mmol / L EDTA, 10 mmol / L Tris-HCl and 0.05 mmol / L phosphoramidite derivative, and the pH value is adjusted to 8.0 with 1 mol / L NaOH solution. The specific phosphoramidite derivative is α-phosphoramidite.

[0124] 2) The bone adsorption solution includes 6.0 mol / L guanidine thiocyanate, 10 mmol / L Tris-HCl and 3 mol / L ethylene glycol, and the pH value is adjusted to 4.0 with acetic acid-sodium acetate buffer solution.

[0125] 3) The magnetic bead suspension contained 9% by weight of silica carboxyl magnetic beads (Suzhou Beaver Biomedical Engineering Co., Ltd.) with a size of 600 nm dispersed in high-purity water.

[0126] 4) The washing liquid I comprises 80% by volume of anhydrous ethanol and 20% by volume of water.

[0127] 5) The washing liquid II includes 80% by volume of anhydrous ethanol and 20% by volume of water.

[0128] 6) The washing liquid III includes 80% by volume of anhydrous ethanol and 20% by volume of water.

[0129] Comparative Example 2 is compared with the reagent materials used in Example 1 of the present invention:

[0130]

[0131] 2. Experimental steps

[0132] 1) The bone powder was prepared in Example 1.

[0133] 2) Take 3g of bone powder in a screw-capped centrifuge tube, add 5mL of bone incubation solution, 200uL of proteinase K solution (20mg / mL), and 50uL of DTT solution (1mol / L), shake and mix, and place in a water bath at 51°C for 6 hours, shaking vigorously 5 times. Then place the centrifuge tube in a centrifuge at 7000rpm for 5 minutes, and transfer the supernatant to another centrifuge tube;

[0134] 3) Add 50 mg of carboxylic acid-based cationic resin to the supernatant, shake and mix, place in a metal bath at 80°C for 20 minutes, and then centrifuge at 7000 rpm for 3 minutes, and transfer the supernatant to another centrifuge tube;

[0135] 4) Add 10 mL of bone adsorption solution to the supernatant, then add 50 uL of magnetic bead suspension, shake and mix, place the centrifuge tube on an automatic flip mixer, flip and mix to adsorb for 30 minutes, immediately put it back on the magnetic stand after adsorption, magnetically absorb for 5 minutes, then discard all the liquid to obtain magnetic beads adsorbed with DNA;

[0136] 5) Add 600uL of washing solution I to the centrifuge tube, shake and mix, immediately put it back on the magnetic stand, magnetically absorb for 2 minutes, and discard all the liquid; then add 800uL of washing solution II, shake and mix, immediately put it back on the magnetic stand, magnetically absorb for 2 minutes, and discard all the liquid; finally, add 800uL of washing solution III, shake and mix, immediately put it back on the magnetic stand, magnetically absorb for 2 minutes, and discard all the liquid;

[0137] 6) Open the centrifuge tube and place it in a 65℃ metal bath to dry for 5 minutes. Then add 20uL of high-purity water to disperse all the magnetic beads into the solution. Close the lid and warm it in a 65℃ metal bath for 10 minutes. After shaking and mixing, immediately put it back on the magnetic rack and magnetically absorb it for 2 minutes. Aspirate the DNA solution and save it in another centrifuge tube.

[0138] The total extraction time was approximately 8 hours.

[0139] The DNA solution obtained in Comparative Example 2 was amplified by PCR using the FastDirect DNA 25A Amplification Kit (Baiteyuan Biotechnology Co., Ltd.), and electrophoresed in an ABI 3500XL instrument. The STR typing spectrum obtained is as follows: Figure 5 As shown, the STR loci are incomplete, 18 alleles among the 25 loci are complete, the typing is inaccurate, and the peak height is basically below 800, indicating that the purification method has a short extraction time and low purification efficiency, and cannot obtain DNA with purity that meets the requirements. The integrity is poor and it is not suitable for extracting trace DNA from old bone samples.

[0140] Comparative Example 3

[0141] 1. Reagent materials:

[0142] 1) Decalcification solution includes 0.5 mol / L EDTA pH 8.0 (Promega)

[0143] 2) The bone incubation solution includes 0.05 wt % sodium dodecyl sulfate, 15 mmol / L EDTA and 10 mmol / L Tris-HCl, and the pH value is adjusted to 8.0 with 1 mol / L NaOH solution.

[0144] 3) The bone adsorption solution includes 6.0 mol / L guanidine thiocyanate, 10 mmol / L Tris-HCl and 3 mol / L ethylene glycol, and the pH value is adjusted to 4.0 with acetic acid-sodium acetate buffer solution.

[0145] 4) The magnetic bead suspension contained 9% by weight of silica carboxyl magnetic beads (Suzhou Beaver Biomedical Engineering Co., Ltd.) with a size of 600 nm dispersed in high-purity water.

[0146] 5) The washing liquid I comprises 80% by volume of anhydrous ethanol and 20% by volume of water.

[0147] 6) The washing liquid II includes 80% by volume of anhydrous ethanol and 20% by volume of water.

[0148] 7) The washing liquid III includes 80% by volume of anhydrous ethanol and 20% by volume of water.

[0149] Comparative Example 3 is compared with the reagent materials used in Example 1 of the present invention:

[0150]

[0151]

[0152] 2. Experimental steps

[0153] 1) The bone powder was prepared in Example 1.

[0154] 2) Take 3 g of bone powder in a screw-cap centrifuge tube, add 5 mL of decalcification solution, shake and mix, place on an automatic flip mixer, decalcify at room temperature for 12 h, then place the centrifuge tube in a centrifuge at 7000 rpm for 5 minutes, and discard all the liquid.

[0155] 3) Add 5 mL of bone incubation solution, 200 uL of proteinase K (20 mg / mL), and 50 uL of DTT (1 mol / L) to the decalcified centrifuge tube, shake and mix, and place in a water bath at 51°C for 6 hours, shaking vigorously 5 times. Then place the centrifuge tube in a centrifuge at 7000 rpm for 5 minutes, and transfer the supernatant to another centrifuge tube.

[0156] 4) Add 10 mL of bone adsorption solution to the supernatant, then add 50 uL of magnetic bead suspension, shake and mix, place the centrifuge tube on an automatic flip mixer, flip and mix for 30 minutes, and immediately put it back on the magnetic stand after adsorption, magnetically absorb for 5 minutes, then discard all the liquid to obtain magnetic beads adsorbed with DNA.

[0157] 5) Add 600uL of washing solution I to the centrifuge tube, shake and mix, immediately put it back on the magnetic stand, magnetically absorb for 2 minutes, and discard all the liquid; then add 800uL of washing solution II, shake and mix, immediately put it back on the magnetic stand, magnetically absorb for 2 minutes, and discard all the liquid; finally, add 800uL of washing solution III, shake and mix, immediately put it back on the magnetic stand, magnetically absorb for 2 minutes, and discard all the liquid;

[0158] 6) Open the centrifuge tube and place it in a 65℃ metal bath to dry for 5 minutes. Then add 20uL of high-purity water to disperse all the magnetic beads into the solution. Close the lid and warm it in a 65℃ metal bath for 10 minutes. After shaking and mixing, immediately put it back on the magnetic rack and magnetically absorb it for 2 minutes. Aspirate the DNA solution and save it in another centrifuge tube.

[0159] The total extraction time was approximately 20 hours.

[0160] The DNA solution obtained in Comparative Example 3 was amplified by PCR using the FastDirect DNA 25A Amplification Kit (Baiteyuan Biotechnology Co., Ltd.), and electrophoresed in an ABI 3500XL instrument. The STR typing spectrum obtained is as follows: Figure 6 As shown, the STR loci are complete and accurately typed, with 25 loci alleles intact, but the peak heights are basically below 1500, indicating that the purification method can obtain DNA with purity that meets the requirements, but its extraction time is long, the sample content is low, and the purification efficiency is low, so it is not suitable for extracting trace DNA from old bone samples.

[0161] Through the above experimental comparison, it is found that the method of the present invention has a better extraction effect. The method of the present invention is easy to operate, does not require separate decalcification pretreatment, avoids sample loss, has high sensitivity in DNA extraction, strong resistance to impurity interference, and high detection rate. It can effectively extract and purify trace DNA (100pg to 250pg) from old bone samples, fully demonstrating the applicability and effectiveness of the method of the present invention, and can provide a more effective basis for case detection and court trials.

[0162] Compared with the prior art, the beneficial effects of the present invention are mainly:

[0163] 1. The present invention provides a method for extracting trace DNA from old bone samples without decalcification, which replaces the traditional EDTA decalcification step and directly introduces a specific metal chelator into the bone incubation solution. Compared with EDTA and its sodium salt, the specific metal chelator has a stronger chelating ability for calcium and magnesium ions, and the specific metal chelator and its complex product are highly water-soluble, not easy to precipitate, and easy to remove, and will not affect the efficiency of magnetic bead adsorption of DNA and subsequent PCR amplification.

[0164] 2. The present invention refers to a method for extracting trace DNA from soil samples disclosed in the Chinese invention patent ZL202010324464.3 of the applicant. A phosphoramidite derivative is also added to the bone incubation solution, so that it preferentially acts on the fixed sites of the phosphate group on the surface of the soil, greatly reducing the adsorption of DNA by the soil, and being able to obtain the incubation product to the maximum extent. The soil impurities are then removed by centrifugation to ensure that there is no adverse effect on the subsequent adsorption of DNA by magnetic beads. The excess phosphoramidite derivative in the reaction system is adsorbed by a carboxylic acid-based cationic resin and then removed by centrifugation. Therefore, minimizing the impact of soil impurities in old bone samples on DNA extraction and recovery is beneficial to improving the efficiency of trace DNA recovery in old bone samples, thereby effectively improving the detection rate of old bone samples with high impurity content.

[0165] 3. In order to eliminate the influence of metal impurities on the extraction effect as much as possible, the present invention can also introduce specific metal chelators with different proportions into the washing solution I system to minimize the metal impurities being brought into the elution system by the magnetic beads, thereby improving the sample purity and reducing the influence of impurities on subsequent PCR amplification.

[0166] Therefore, the present invention provides a method for extracting trace DNA from old bone samples, which can achieve effective extraction of DNA from old bone samples, eliminate the need for a separate decalcification step, simplify the operation process, shorten the extraction time, has strong resistance to impurity inhibition, and can effectively improve the success rate of extracting trace DNA from old bone samples.

[0167] The present invention is a method for simplifying the extraction of old bone samples, avoiding sample loss caused by a single decalcification step, enhancing the anti-interference ability of soil impurities, thereby improving the detection rate of old bone samples, and has broad application prospects.

[0168] In summary, the method for extracting trace DNA from old bone samples without decalcification of the present invention can effectively extract and purify trace DNA from old bone samples, does not require separate decalcification pretreatment, and avoids sample loss. The extracted DNA can be used for forensic identification, and can provide a more effective basis for case detection and court trials. It is ingeniously designed, uniquely conceived, and easy to operate. It eliminates the need for a separate decalcification step, simplifies the operating process, shortens the extraction time, and is suitable for large-scale promotion and application.

[0169] In this specification, the present invention has been described with reference to specific embodiments thereof. However, it is apparent that various modifications and variations may be made without departing from the spirit and scope of the present invention. Therefore, the description should be regarded as illustrative rather than restrictive.

Claims

1. A method for extracting trace amounts of DNA from old bone samples without decalcification, characterized in that: The following steps are involved: (1) Incubation: Mix bone powder, bone incubation solution, proteinase K solution and DTT solution obtained from old bone samples and incubate them, shake them vigorously several times during the process, and centrifuge to obtain a first supernatant, wherein the bone incubation solution comprises 0.05wt% to 0.2wt% of sodium dodecyl sulfate, 5mmol / L to 15mmol / L of EDTA, 10mmol / L to 40mmol / L of Tris-HCl, 0.1mol / L to 0.5mol / L of a specific metal chelator and 0.05mmol / L to 0.3mmol / L of an aminophosphoric acid ester derivative, the pH value of the bone incubation solution is 7.5 to 8.5, and the specific metal chelator is selected from one or more of tetrasodium iminodisuccinate, tetrasodium glutamate diacetate, trisodium N,N-dicarboxymethylalanine, sodium gluconate and sodium ethylenediamine di-o-phenylacetate; (2) adding a carboxylic acid-based cationic resin to the first supernatant, mixing well, placing in a metal bath, and centrifuging to obtain a second supernatant; (3) adding bone adsorption liquid and magnetic bead suspension to the second supernatant, turning over and mixing for adsorption, and then magnetically aspirating to discard all liquid to obtain magnetic beads adsorbed with DNA; (4) Washing and eluting the magnetic beads adsorbed with DNA to obtain a DNA solution of the old bone sample.

2. The method for extracting trace amounts of DNA from old bone samples without decalcification according to claim 1, characterized in that: In the step (1), the bone powder is 1g to 3g, the bone incubation solution is 3mL to 5mL, the proteinase K solution is 100uL to 300uL, the concentration of the proteinase K solution is 20mg / mL, the DTT solution is 20uL to 60uL, the concentration of the DTT solution is 1mol / L, the incubation temperature is 37°C to 56°C, the incubation time is 1h to 12h, the number of vigorous shaking is 5 to 8 times, the centrifugal speed is 7000rpm to 10000rpm, and the centrifugal time is 5 minutes to 8 minutes.

3. The method for extracting trace amounts of DNA from old bone samples without decalcification according to claim 1, characterized in that: In the step (1), the amino phosphoroester derivative is selected from one or more of α-amino phosphoroester, β-amino phosphoroester and γ-amino phosphoroester, wherein the amino group is an unsubstituted amino group, a monosubstituted amino group or a disubstituted amino group, the substituent of the amino group is an aliphatic group, and the amino phosphoroester is a fatty alcohol phosphate with a length of less than 5 carbon atoms.

4. The method for extracting trace DNA from old bone samples without decalcification according to claim 1, characterized in that: In the step (2), the carboxylic acid-based cationic resin is 10 mg to 50 mg, the temperature of the metal bath is 60° C. to 90° C., the time of the metal bath is 10 minutes to 20 minutes, the speed of the centrifuge is 7000 rpm to 10000 rpm, and the time of the centrifuge is 2 minutes to 4 minutes.

5. The method for extracting trace DNA from old bone samples without decalcification according to claim 1, characterized in that: In step (3), the bone adsorption solution is 8 mL to 15 mL, and the bone adsorption solution includes 3.0 mol / L to 6.0 mol / L of guanidine isothiocyanate, 10 mmol / L to 40 mmol / L of Tris-HCl and 1.5 mol / L to 3 mol / L of ethylene glycol. The pH value of the bone adsorption solution is 3 to 5, the magnetic bead suspension is 30 uL to 60 uL, the flipping mixing adsorption time is 20 minutes to 30 minutes, and the magnetic attraction time is 5 minutes to 10 minutes.

6. The method for extracting trace amounts of DNA from old bone samples without decalcification according to claim 1, characterized in that: In step (3), the magnetic bead suspension includes 9 wt% of silica magnetic composite particles with a size of 100 nm to 2000 nm, wherein the silica magnetic composite particles are cross-linked bodies formed by cross-linking two or more ferrite-silica core-shell nanoparticles, and the surface of the silica magnetic composite particles has multiple nanoscale protrusions.

7. The method for extracting trace DNA from old bone samples without decalcification according to claim 1, characterized in that: In the step (4), the specific steps of washing include: using washing solution I to perform the first washing and magnetic absorption on the magnetic beads adsorbed with DNA, and aspirating all liquid, using washing solution II to perform the second washing and magnetic absorption, and aspirating all liquid, and using washing solution III to perform the third washing and magnetic absorption, and aspirating all liquid.

8. The method for extracting trace amounts of DNA from old bone samples without decalcification according to claim 7, characterized in that: In the step (4), the washing solution I includes 2mol / L to 5mol / L of guanidine thiocyanate, 5mmol / L to 25mmol / L of Tris-HCl and 10mmol / L to 50mmol / L of a specific metal chelator, and the amount of the washing solution I used is 300uL to 700uL. The washing solution II includes 2mol / L of sodium chloride, 50% by volume of anhydrous ethanol, 20% by volume of isopropanol and 30% by volume of water, and the amount of the washing solution II used is 600uL to 1000uL. The washing solution III includes 50% by volume of anhydrous ethanol and 50% by volume of isopropanol, and the amount of the washing solution III used is 600uL to 1000uL.

9. The method for extracting trace amounts of DNA from old bone samples without decalcification according to claim 1, characterized in that: In the step (4), the specific steps of eluting include: drying the magnetic beads adsorbed with DNA, adding water and mixing to dissolve them, and the liquid obtained after mixing and magnetic absorption is the DNA solution.

10. The method for extracting trace amounts of DNA from old bone samples without decalcification according to claim 9, characterized in that: In the step (4), the drying is performed in a 65°C metal bath for 5 to 10 minutes, the amount of water used is 20uL to 100uL, the dissolution is performed in a 65°C metal bath for 10 minutes, and the magnetic attraction time is 2 minutes.

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