CN114736898B - Method for extracting free edge nuclear DNA of nail and application - Google Patents
Method for extracting free edge nuclear DNA of nail and application Download PDFInfo
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- CN114736898B CN114736898B CN202210541747.2A CN202210541747A CN114736898B CN 114736898 B CN114736898 B CN 114736898B CN 202210541747 A CN202210541747 A CN 202210541747A CN 114736898 B CN114736898 B CN 114736898B
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- 238000012360 testing method Methods 0.000 claims description 9
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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Abstract
The invention discloses a method for extracting free edge nuclear DNA of nails and application thereof, which comprises the steps of cutting nails close to finger tips into small blocks, grinding the small blocks into powder in a cryo-grinder pre-cooled to-70 ℃ to-60 ℃ in advance, and centrifuging at high speed; adding digestive juice to digest the nails; centrifuging at high speed, and collecting supernatant. The method for extracting the free edge nuclear DNA of the nail can greatly increase the success rate of extracting the free edge nuclear DNA of the nail, the STR parting map of the extracted free edge nuclear DNA is qualified, the available probability is greatly improved, and the re-extraction rate and the repeated sampling probability are reduced. The whole extraction process is simplified, the harm to the body caused in the use process of the organic reagent is reduced, and the cost is low.
Description
Technical Field
The invention belongs to the technical field of biology, and in particular relates to a method for extracting nuclear DNA from free edges of nails and application thereof.
Background
Forensic physical evidence is a branch science of forensic science, is a science for researching and applying life science technology to solve the problem of biological detection material identification related to human body in cases, and is to prove the fact of the cases by the components and characteristics of the biological detection material, wherein the biological detection material generally comprises blood, semen, vaginal secretion, saliva, amniotic fluid and marks thereof; hair, nails, bone, and teeth; various tissues, organs and fragments of human body, etc. The main task of forensic physical evidence inspection is to solve the personal identification and the right-of-ownership identification problems in judicial practice.
With the increasing depth of DNA genetic markers, DNA typing has become a major tool in forensic physical evidence testing. Personal identification is performed by DNA typing without determining the whole genome sequence. The advantage of DNA typing is also that the same result can be obtained from any body fluid or tissue containing cells, and thus, in the actual identification work, the selection of the test material is good or bad. The nail is often the preferred test material for the identified person because of its non-invasive, concealed, easy to obtain, etc. And the nails are easy to store and transport, and can be selected as identification materials for patients with serious illness or blood transfusion history. The nail has strong anti-putrefaction, so the nail can be used as a cadaver inspection material.
The nail is formed by keratinization of epidermis cells, and is composed of multiple layers of keratinocytes which are firmly connected, and the cells are rich in keratin, and comprise three parts of free edge, nail body and nail root. In the case of some criminal cases, the DNA test of nails requires pulling out the entire nail and digesting the nail body or root portion of the nail to obtain a satisfactory DNA test result. However, in the field of paternity test, it is not practical to extract the whole nail for test, and in general, only the free edge of the nail can be obtained for test. The lower part of the free edge of the nail is not supported, and the nail cells lack nutrition supply of the nail bed, so that compared with other parts of the nail, the cytokeratosis degree is higher, the cell nucleus is degenerated, and the nuclear DNA is degraded more seriously, so that the content is very low, the DNA extraction difficulty is very high, and meanwhile, the DNA extraction difficulty is increased because the free edge of the nail has compact structure.
At present, the nail DNA is generally extracted by adopting an organic method combined with a Chelex-100 extraction method, a Chelex-100 combined adsorption column method or a magnetic bead method, and the pretreatment operation steps of the methods are various: generally, a proper amount of nails are put into a high-speed centrifuge tube, 1.5mL of 10% SDS is added, shaking and soaking are carried out for about 30min, deionized water is used for repeatedly soaking and washing for 2-3 times, double distilled water is used for rinsing for 2-3 times, and after drying, ophthalmic scissors are used for cutting into small blocks. The series of operations have the disadvantages of long time consumption, easy pollution, difficulty in extracting enough DNA, poor experimental amplification effect and difficulty in electrophoresis of a complete map. And organic solvents can cause a degree of harm to the body. DNA extraction kits for nail free edges are also available on the market, but are expensive and costly.
Therefore, DNA extraction is difficult due to the special structure of the free edge of the nail, and the DNA extraction is not currently widely used as a preferred identification material for forensic material science. It is very significant to explore a simple, accurate and low cost method for extracting DNA.
Disclosure of Invention
Based on this, it is an object of the present invention to provide a method for extracting free edge nuclear DNA of nails, which can greatly improve the success rate and quality of extracting free edge nuclear DNA, and is simple, quick, and does not require the use of an organic solvent.
The specific technical scheme for realizing the aim of the invention comprises the following steps:
a method of extracting nail free edge nuclear DNA comprising the steps of:
(1) Cutting the nails near the finger tips into small blocks, performing cold grinding into powder in a cold grinder pre-cooled to-70 ℃ to-60 ℃ in advance, and performing high-speed centrifugation;
(2) Adding digestive juice to digest the nails;
(3) Centrifuging at high speed, and collecting supernatant.
In some embodiments, the cryomill in step (1) is pre-chilled to-66 ℃ to-64 ℃ in advance.
In some of these embodiments, the parameters of the freeze-milling in step (1) include: operating frequency: 70 Hz-80 Hz; run time: 50 s-70 s; stop time: 10 s-20 s; cycle times: 6 to 10 times.
In some of these embodiments, the parameters of the freeze-grinding include: operating frequency: 74 Hz-76 Hz; run time: 55 s-65 s; stop time: 14 s-16 s; cycle times: 7 times to 9 times.
In some of these embodiments, the digesting in step (2) comprises: the primary digestion is carried out for 6 to 24 hours at 54 to 58 ℃ and then the secondary digestion is carried out for 6 to 10 minutes at 100 ℃. Preferably, the primary digestion is carried out at 56℃for 6 to 24 hours and then the secondary digestion is carried out at 100℃for 8 minutes.
In some embodiments, after adding the digestive juice in the step (2), shaking, uniformly mixing and centrifuging for 8-12 s, and then performing primary digestion; and/or after primary digestion, shaking, uniformly mixing and centrifuging for 8-12 s, and then performing secondary digestion.
In some embodiments, the digestive juice of step (3) comprises: 3 to 7 weight percent of Chelex-100 suspension 350 mu L to 450 mu L, 8mg/mL to 12mg/mL of proteinase K solution 8 mu L to 12 mu L, 0.8mol/L to 1.2mol/L of dithiothreitol 8 mu L to 12 mu L.
In some of these embodiments, in step (1), the nail surface is scraped clean with a surgical blade prior to cutting the nail into small pieces.
In some embodiments, the high-speed centrifugation in step (1) is performed at a speed of 10000r/min to 14000r/min for 2min to 4min;
in some embodiments, the high speed centrifugation in step (3) is performed at a speed of 10000r/min to 14000r/min for a period of 2min to 4min.
The invention also provides the free edge nuclear DNA of the nail extracted by the method.
The invention also provides application of the free edge nuclear DNA in forensic physical evidence inspection.
Compared with the prior art, the invention has the following beneficial effects:
according to the method for extracting the free edge nuclear DNA of the nail, the free edge of the nail is frozen and ground under the freezing condition, the temperature and the parameters of the frozen and ground are precisely controlled, and the frozen and ground powder is digested, so that the success rate of extracting the free edge nuclear DNA of the nail can be greatly increased, the probability of qualified and usable STR parting map is greatly improved, and the re-extraction rate and the probability of repeated sampling are reduced. In addition, the whole extraction process is simplified, a plurality of complicated pretreatment steps are omitted, the use of an organic solvent is avoided, the harm to the body in the use process of the organic reagent is reduced, and meanwhile, the cost is low.
Drawings
FIG. 1 is a map of the STR part locus of the free nuclear DNA of the nail extracted in example 1 of the present invention.
FIG. 2 is a map of the STR portion locus of the free nuclear DNA of the nail extracted in example 2 of the present invention.
FIG. 3 is a map of the STR portion locus of the free nuclear DNA of the nail extracted in example 3 of the present invention.
FIG. 4 is a map of the STR portion locus of the free nuclear DNA of the nail extracted in comparative example 1 of the present invention.
FIG. 5 is a map of STR part locus of the free nuclear DNA of nail extracted in comparative example 2 of the present invention.
FIG. 6 is a map of STR part locus of the free nuclear DNA of nail extracted in comparative example 3 of the present invention.
FIG. 7 is a map of STR part locus of the free nuclear DNA of nail extracted in comparative example 4 of the present invention.
FIG. 8 is a map of STR part locus of the free nuclear DNA of nail extracted in comparative example 5 of the present invention.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
In one aspect of the present invention, there is provided a method for extracting DNA from the free edge nucleus of a nail, comprising the steps of:
(1) Selecting 3 mg-10 mg of nails which are as close to the finger tips as possible, and scraping the surfaces of the nails by using a surgical blade to prevent carrying gene pollution and inhibiting non-gene impurities;
(2) Cutting the nails into small pieces, placing the small pieces into an EP (EP) tube special for grinding, and using a cryo-grinder (note that the cryo-grinder is started to be in a refrigerating mode before the experiment starts, so that the temperature of the grinding environment is reduced to-70 ℃ -60 ℃), grinding the nails into powder, wherein parameters are set as follows: operating frequency: 70 Hz-80 Hz; run time: 50 s-70 s; stop time: 10 s-20 s; cycle times: and 6 to 10 times (the operation parameters can be determined according to the actual situation, and the cycle times can be increased when the grinding effect is found to be poor). Preferably, the parameters of the cryo-mill are set as follows: operating frequency: 75Hz; run time: 60S; stop time: 15S; cycle times: 8. nail is used as keratinized hard tissue, the tissue is compact, the freezing can increase the brittleness of the nail to make the grinding more sufficient, and simultaneously, the nail can be directly frozen to a cell layer after grinding and crushing, so that the keratinized cell membrane is fully destroyed and depolymerized, and the cell lysis and digestion are more thorough.
(3) Placing the EP pipe filled with the nail powder into a high-speed centrifuge, and centrifuging for 2-4 min at 10000 r/min-14000 r/min to enable the nail powder on the pipe wall of the EP pipe to be centrifuged to the bottom as much as possible; preferably, the rotating speed of the high-speed centrifugal machine is 12000r/min, and the centrifugal time is 3min;
(4) Adding 350-450 mu L of Chelex-100 suspension with the mass concentration of 3-7%, 8-12 mg/mL of proteinase K solution, 8-12 mu L of proteinase K solution, 0.8-1.2 mol/L of DTT (dithiothreitol) and 8-12 mu L of DTT (dithiothreitol), shaking and uniformly mixing, putting into a small-sized centrifuge, centrifuging for 8-12 s, and throwing the liquid on the tube wall to the bottom; placing the mixture into a constant temperature instrument at 54-58 ℃ for heat preservation, incubation and digestion for more than 6 hours (preferably, incubation is carried out overnight, and the effect is better);
(5) After shaking and mixing uniformly, putting the mixture into a small-sized centrifuge for centrifugation for 8-12 s, putting the mixture into a thermostat at 100 ℃, and incubating the mixture for 6-10 min again;
(6) And (3) after shaking and mixing uniformly, centrifuging for 2-4 min (preferably, the rotating speed of the high-speed centrifuge is 12000r/min and the centrifuging time is 3 min) in a high-speed centrifuge, and taking supernatant to obtain the high-speed centrifuge.
The invention is described in detail below with reference to the drawings and the specific embodiments.
Example 1A method for extracting DNA from free edge nuclei of nails
The method for extracting the free edge nuclear DNA of the nail comprises the following steps:
(1) About 7mg of the free edge sample of the nail (sample number 2022-J18-Z) of volunteer No. 1 near the finger tip was selected and the nail surface was scraped clean with a surgical blade;
(2) Firstly, starting a refrigeration mode of a cryo-grinder, cutting nails into small blocks, putting the small blocks into a 2.0mL special EP pipe for grinding, adding three grinding steel balls, starting the cryo-grinder when the cryo-grinder is lowered to-65 ℃, grinding the nails into powder, and setting parameters as follows: operating frequency: 75Hz; run time: 60s; stop time: 15s; cycle times: 8, 8;
(3) Placing the EP pipe filled with the nail powder into a high-speed centrifuge, centrifuging for 3min at 12000r/min to centrifuge the nail powder on the wall of the EP pipe to the bottom as much as possible, and removing the grinding steel balls;
(4) Adding 400 mu L of 5% Chelex-100 suspension, 10 mu L of 10mg/mL proteinase K solution and 10 mu L of 1.0mol/L DTT (dithiothreitol), shaking, mixing, centrifuging in a small-sized centrifuge for 10s, and throwing the liquid on the pipe wall to the bottom; placing the mixture in a thermostat at 56 ℃ for heat preservation, incubation and digestion overnight;
(5) Shaking and uniformly mixing the digested sample, putting the sample into a small-sized centrifuge for centrifugation for 10s, putting the sample into a thermostat at 100 ℃, and incubating for 8min again;
(6) Shaking and uniformly mixing the nail sample, and centrifuging in a high-speed centrifuge for 3min at 12000 r/min;
(7) Transferring the supernatant after centrifugation into another clean EP tube to obtain the free edge nuclear DNA of the nail.
1. Mu.L of the DNA extracted in this example was taken and fluorescently labeled China was used TM Platinum PCR amplification kit for multiplex STR locus amplification (using the methods described in the kit instructions); the amplified products were electrophoresed using an ABI 3500XL genetic analyzer in the United states and usedAnd (3) carrying out data analysis by the ID-X software, and deriving an STR map.
As shown in figure 1, under the condition that the allelic typing control (Ladder and positive control genotyping sites) is accurate, the peak height of each allelic typing of the map reaches more than 100bp, and most allelic typing is relatively balanced. The specific typing results are shown in Table 1.
TABLE 1 STR locus typing results
| Sample numbering | D3S1358 | vWA | D16S539 | CSF1PO | TPOX | D8S1179 | D21S11 | D18S51 |
| 2022-J18-Z | 15,16 | 14,19 | 9,11 | 12 | 8 | 13,15 | 29 | 15 |
| Sample numbering | PentaE | D2S441 | D19S433 | TH01 | FGA | D22S1045 | D5S818 | D13S317 |
| 2022-J18-Z | 12,17 | 10,14 | 13,15.2 | 9,10 | 23,26 | 11,16 | 11,13 | 8 |
| Sample numbering | D7S820 | D6S1043 | D10S1248 | D1S1656 | D12S391 | D2S1338 | PentaD | AMEL |
| 2022-J18-Z | 11,12 | 11 | 16 | 11,16 | 17,19 | 20,22 | 9,12 | X,Y |
The results of FIG. 1 and Table 1 show that the method of this example was used to successfully extract DNA from the free edge nuclei of nails with good quality.
Example 2A method of extracting DNA from the free edge nucleus of nails
The method for extracting the free edge nuclear DNA of the nail comprises the following steps:
(1) About 4mg of a free edge sample of the nail near the finger tip (sample number: 2021-J191-Z2) of volunteer No. 2 was selected, and the nail surface was scraped clean with a surgical blade;
(2) Firstly, starting a refrigeration mode of a cryo-grinder, cutting nails into small blocks, putting the small blocks into a 2.0mL special EP pipe for grinding, adding three grinding steel balls, starting the cryo-grinder when the cryo-grinder is lowered to-65 ℃, grinding the nails into powder, and setting parameters as follows: operating frequency: 75Hz; run time: 60s; stop time: 15s; cycle times: 7, preparing a base material;
(3) Placing the EP pipe filled with the nail powder into a high-speed centrifuge, centrifuging for 3min at 12000r/min to centrifuge the nail powder on the wall of the EP pipe to the bottom as much as possible, and removing the grinding steel balls;
(4) Adding 400 mu L of 5% Chelex-100 suspension, 10 mu L of 10mg/mL proteinase K solution and 10 mu L of 1.0mol/L DTT (dithiothreitol), shaking, mixing, centrifuging in a small-sized centrifuge for 10s, and throwing the liquid on the pipe wall to the bottom; placing the mixture in a thermostat at 56 ℃ for heat preservation, incubation and digestion overnight;
(5) Shaking and uniformly mixing the digested sample, putting the sample into a small-sized centrifuge for centrifugation for 10s, putting the sample into a thermostat at 100 ℃, and incubating for 8min again;
(6) Shaking and uniformly mixing the nail sample, and centrifuging in a high-speed centrifuge for 3min at 12000 r/min;
(7) Transferring the supernatant after centrifugation into another clean EP tube to obtain the free edge nuclear DNA of the nail.
As shown in figure 2, under the condition that the allelic typing control (Ladder and positive control genotyping sites) is accurate, the peak height of each allelic typing of the map reaches more than 100bp, and most allelic typing is relatively balanced. The specific typing results are shown in Table 2.
TABLE 2 STR locus typing results
| Sample numbering | D3S1358 | vWA | D16S539 | CSF1PO | TPOX | D8S1179 | D21S11 | D18S51 |
| 2021-J191-Z2 | 15,16 | 14 | 9 | 11,12 | 11 | 14,15 | 30,33.2 | 13,18 |
| Sample numbering | PentaE | D2S441 | D19S433 | TH01 | FGA | D22S1045 | D5S818 | D13S317 |
| 2021-J191-Z2 | 18.4,21 | 10,11 | 13,14 | 7 | 26 | 15,17 | 11,13 | 8 |
| Sample numbering | D7S820 | D6S1043 | D10S1248 | D1S1656 | D12S391 | D2S1338 | PentaD | AMEL |
| 2021-J191-Z2 | 11 | 14,19 | 14 | 16 | 18,20 | 20,25 | 9,12 | X,Y |
The results of FIG. 2 and Table 2 show that the method of this example was used to successfully extract DNA from the free edge nuclei of nails with good quality.
Example 3A method of extracting DNA from the free edge nucleus of nails
The method for extracting the free edge nuclear DNA of the nail comprises the following steps:
(1) About 10mg of a free edge sample of the nail near the finger tip (sample number: 2021-J216-F) of volunteer No. 3 was selected, and the nail surface was scraped clean with a surgical blade;
(2) Firstly, starting a refrigeration mode of a cryo-grinder, cutting nails into small blocks, putting the small blocks into a 2.0mL special EP pipe for grinding, adding three grinding steel balls, starting the cryo-grinder when the cryo-grinder is lowered to-65 ℃, grinding the nails into powder, and setting parameters as follows: operating frequency: 75Hz; run time: 60s; stop time: 15s; cycle times: 6, preparing a base material;
(3) Placing the EP pipe filled with the nail powder into a high-speed centrifuge, centrifuging for 3min at 12000r/min to centrifuge the nail powder on the wall of the EP pipe to the bottom as much as possible, and removing the grinding steel balls;
(4) Adding 400 mu L of 5% Chelex-100 suspension, 10 mu L of 10mg/mL proteinase K solution and 10 mu L of 1.0mol/L DTT (dithiothreitol), shaking, mixing, centrifuging in a small-sized centrifuge for 10s, and throwing the liquid on the pipe wall to the bottom; placing the mixture in a thermostat at 56 ℃ for heat preservation, incubation and digestion overnight;
(5) Shaking and uniformly mixing the digested sample, putting the sample into a small-sized centrifuge for centrifugation for 10s, putting the sample into a thermostat at 100 ℃, and incubating for 8min again;
(6) Shaking and uniformly mixing the nail sample, and centrifuging in a high-speed centrifuge for 3min at 12000 r/min;
(7) Transferring the supernatant after centrifugation into another clean EP tube to obtain the free edge nuclear DNA of the nail.
As shown in figure 3, under the condition that the allelic typing control (Ladder and positive control genotyping sites) is accurate, the peak height of each allelic typing of the map reaches more than 100bp, and most allelic typing is relatively balanced. The specific typing results are shown in Table 3.
TABLE 3 STR locus typing results
The results of FIG. 3 and Table 3 show that the method of this example was used to successfully extract DNA from the free edge of nails, and the pattern was acceptable and of good quality.
Comparative example 1A method for extracting DNA from free edge nucleus of nail
A method for extracting free edge nuclear DNA of nails of this comparative example comprises the steps of:
(1) About 7mg of the free edge sample of the nail (sample number 2022-J18-Z) of volunteer No. 1 near the finger tip was selected and the nail surface was scraped clean with a surgical blade;
(2) Cutting the nails into small blocks, putting the small blocks into a 2.0mL EP tube, adding 400 mu L of 5% Chelex-100 suspension, 10 mu L of 10mg/mL proteinase K solution and 10 mu L of 1.0mol/L DTT (dithiothreitol), shaking and mixing uniformly, putting into a small centrifuge, centrifuging for 10s, and throwing the liquid on the tube wall to the bottom; placing the mixture in a thermostat at 56 ℃ for heat preservation, incubation and digestion overnight;
(3) Shaking and uniformly mixing the digested sample, putting the sample into a small-sized centrifuge for centrifugation for 10s, putting the sample into a thermostat at 100 ℃, and incubating for 8min again;
(4) Shaking and uniformly mixing the nail sample, and centrifuging in a high-speed centrifuge for 3min at 12000 r/min;
(5) Transferring the supernatant after centrifugation into another clean EP tube to obtain DNA.
As shown in FIG. 4, in the case of the accurate allele-typing control (Ladder and positive control genotyping site), the allele-typing peak height of the STR portion was not uniform, the peak was too low, and the allele-typing deletion occurred. The specific typing results are shown in Table 4.
TABLE 4 STR locus typing results
Note that: in the table "/" indicates site deletion
The results of fig. 4 and table 4 show that when the nail-free edge sample is directly digested without freeze-grinding, the effect of cell lysis is affected, the nail-free edge nuclear DNA typing pattern is failed, and DNA extraction fails.
Comparative example 2A method for extracting DNA from free edge nucleus of nail
A method for extracting free edge nuclear DNA of nails of this comparative example comprises the steps of:
(1) About 10mg of a free edge sample of the nail near the finger tip (sample number: 2021-J137-F) of volunteer No. 6 was selected, and the nail surface was scraped clean with a surgical blade;
(2) Firstly, starting a refrigeration mode of a cryo-grinder, cutting nails into small blocks, putting the small blocks into a 2.0mL special EP pipe for grinding, adding three grinding steel balls, starting the cryo-grinder when the cryo-grinder is lowered to-65 ℃, grinding the nails into powder, and setting parameters as follows: operating frequency: 35Hz; run time: 60s; stop time: 15s; cycle times: 2;
(3) Placing the EP pipe filled with the nail powder into a high-speed centrifuge, centrifuging for 3min at 12000r/min to centrifuge the nail powder on the wall of the EP pipe to the bottom as much as possible, and removing the grinding steel balls;
(4) Adding 400 mu L of 5% Chelex-100 suspension, 10 mu L of 10mg/mL proteinase K solution and 10 mu L of 1.0mol/L DTT (dithiothreitol), shaking, mixing, centrifuging in a small-sized centrifuge for 10s, and throwing the liquid on the pipe wall to the bottom; placing the mixture in a thermostat at 56 ℃ for heat preservation, incubation and digestion overnight;
(5) Shaking and uniformly mixing the digested sample, putting the sample into a small-sized centrifuge for centrifugation for 10s, putting the sample into a thermostat at 100 ℃, and incubating for 8min again;
(6) Shaking and uniformly mixing the nail sample, and centrifuging in a high-speed centrifuge for 3min at 12000 r/min;
(7) Transferring the supernatant after centrifugation into another clean EP tube to obtain the free edge nuclear DNA of the nail.
As shown in FIG. 5, in the case of the exact allele-typing control (Ladder and positive control genotyping site), the allele-typing peak heights of the STR portion were not uniform, the peak was too low, and the allele-typing deletion occurred. The specific typing results are shown in Table 5.
TABLE 5 STR locus typing results
| Sample numbering | D3S1358 | vWA | D16S539 | CSF1PO | TPOX | D8S1179 | D21S11 | D18S51 |
| 2021-J137-F | 15,16 | 14,16 | / | / | 11 | 11,15 | 30,31.2 | / |
| Sample numbering | PentaE | D2S441 | D19S433 | TH01 | FGA | D22S1045 | D5S818 | D13S317 |
| 2021-J137-F | / | 10,14 | 14,15.2 | 6,7 | 24,26 | 15 | 11,12 | 12 |
| Sample numbering | D7S820 | D6S1043 | D10S1248 | D1S1656 | D12S391 | D2S1338 | PentaD | AMEL |
| 2021-J137-F | / | / | 13,15 | 15,16 | 18,20 | 20 | / | X,Y |
Note that: in the table "/" indicates site deletion
The results of FIGS. 5 and 5 show that when the nail free edge sample is subjected to freeze-polishing, the polishing frequency is low, and the polishing time is short, insufficient polishing is caused, so that the nail free edge nuclear DNA typing pattern is failed, and DNA extraction is failed.
Comparative example 3A method for extracting DNA from free edge nucleus of nail
A method for extracting free edge nuclear DNA of nails of this comparative example comprises the steps of:
(1) About 7mg of a free edge sample of the nail near the finger tip (sample number: 2021-J179-Z) of volunteer No. 4 was selected and the nail surface was scraped clean with a surgical blade;
(2) Cutting the nails into small pieces, putting the small pieces into a grinding pot for grinding for about 60 minutes, and filling the ground nail scraps into a 2.0mLEP tube;
(3) Adding 400 mu L of 5% Chelex-100 suspension, 10 mu L of 10mg/mL proteinase K solution and 10 mu L of 1.0mol/L DTT (dithiothreitol), shaking, mixing, centrifuging in a small-sized centrifuge for 10s, and throwing the liquid on the pipe wall to the bottom; placing the mixture in a thermostat at 56 ℃ for heat preservation, incubation and digestion overnight;
(4) Shaking and uniformly mixing the digested sample, putting the sample into a small-sized centrifuge for centrifugation for 10s, putting the sample into a thermostat at 100 ℃, and incubating for 8min again;
(5) Shaking and uniformly mixing the nail sample, and centrifuging in a high-speed centrifuge for 3min at 12000 r/min;
(6) Transferring the supernatant after centrifugation into another clean EP tube to obtain the free edge nuclear DNA of the nail.
As shown in FIG. 6, in the case of the accurate allele-typing control (Ladder and positive control genotyping site), the allele-typing peak height of the STR portion was not uniform, the peak was too low, and the allele-typing deletion occurred. The specific typing results are shown in Table 6.
TABLE 6 STR locus typing results
| Sample numbering | D3S1358 | vWA | D16S539 | CSF1PO | TPOX | D8S1179 | D21S11 | D18S51 |
| 2021-J179-Z | 15 | 14 | 9 | 11 | 8,9 | 13,15 | / | / |
| Sample numbering | PentaE | D2S441 | D19S433 | TH01 | FGA | D22S1045 | D5S818 | D13S317 |
| 2021-J179-Z | 17,18 | 12,14 | 14,14.2 | 9,10 | 23,24 | 11,15 | / | / |
| Sample numbering | D7S820 | D6S1043 | D10S1248 | D1S1656 | D12S391 | D2S1338 | PentaD | AMEL |
| 2021-J179-Z | 11 | / | 13,16 | / | 19,20 | 18,20 | / | X,Y |
Note that: in the table "/" indicates site deletion
The results of fig. 6 and table 6 show that when the nail free edge sample is not subjected to freezing operation, and is directly ground by a grinding pot and then subjected to digestion treatment, the effect of cell lysis is affected, the nail free edge nuclear DNA typing pattern is not qualified, and DNA extraction fails.
Comparative example 4A method for extracting DNA from free edge nucleus of nail
The method for extracting the free edge nuclear DNA of the nail of the comparative example adopts the existing DNA extraction method and specifically comprises the following steps:
(1) About 7mg of a nail free edge sample (sample number: 2022-J118-Z2) of a volunteer No. 5 near the finger tip is selected, nails are placed into a 2.0mLEP tube, 1.5mL of 10% SDS is added, the mixture is placed on a MicroBio shaking apparatus for shaking and soaking for 30min at maximum speed, deionized water is used for repeatedly soaking and washing for 2-3 times, double distilled water is used for rinsing for 2-3 times, and after drying, the nails are cut into small blocks and placed into the 2.0mLEP tube.
(2) Adding 400ul of STE buffer solution, 40 mu L of 10mg/mL proteinase K solution, 40 mu L of 1.0mol/L DTT and 100 mu L of 10% SDS, shaking and mixing uniformly, putting into a small-sized centrifuge for centrifugation for 10s, throwing the liquid on the pipe wall to the bottom, and putting into a constant temperature instrument at 56 ℃ for incubation and digestion overnight.
(3) The above liquid was transferred to an EP tube with an equal volume of organic solvent (saturated phenol: chloroform: isoamyl alcohol=25:24:1), mixed well, shaken, and centrifuged at 13,000rpm for 3min.
(4) Transferring the supernatant to another clean centrifuge tube, adding 2.5 times of cold absolute ethyl alcohol, freezing and preserving for 10min, centrifuging at 13,000rpm for 5min, discarding the supernatant, and airing the residual ethyl alcohol at room temperature;
(5) Adding 200 mu L of 5% Chelex-100 suspension into the dried DNA, putting into a thermostat at 56 ℃ for digestion for 6 hours, taking out, shaking vigorously for 5-10 seconds, and centrifuging at 13,000rpm for 3 minutes;
(6) Transferring the supernatant after centrifugation into another clean EP tube to obtain the free edge nuclear DNA of the nail.
As shown in FIG. 7, when the allele parting control (Ladder and positive control genotyping sites) is accurate, the peak height of each allele parting of the map reaches more than 100bp, and most of the allele parting is relatively balanced. The specific typing results are shown in Table 7.
TABLE 7 STR locus typing results
| Sample numbering | D3S1358 | vWA | D16S539 | CSF1PO | TPOX | D8S1179 | D21S11 | D18S51 |
| 2022-J118-Z2 | 17 | 16,19 | 9,11 | 13,14 | 8,11 | 15,16 | 30,33.2 | 14,18 |
| Sample numbering | PentaE | D2S441 | D19S433 | TH01 | FGA | D22S1045 | D5S818 | D13S317 |
| 2022-J118-Z2 | 10,16 | 11,14 | 14,14.2 | 9 | 23 | 15 | 11 | 11 |
| Sample numbering | D7S820 | D6S1043 | D10S1248 | D1S1656 | D12S391 | D2S1338 | PentaD | AMEL |
| 2022-J118-Z2 | 11,12 | 12,19 | 13,15 | 11,12 | 22 | 17,24 | 8,11 | X,Y |
The results of FIG. 7 and Table 7 show that the method of this comparative example (organic method combined with Chelex-100 extraction method) was used to successfully extract DNA from the free edge of nail, and the pattern was acceptable and usable, but the DNA extraction process was cumbersome and time-consuming.
Comparative example 5A method of extracting DNA from free edge nuclei of nails
A method for extracting free edge nuclear DNA of nails of this comparative example comprises the steps of:
(1) About 8mg of a free edge sample of the nail near the finger tip (sample number: 2021-J221-F) of volunteer No. 7 was selected and the nail surface was scraped clean with a surgical blade;
(2) Firstly, starting a refrigeration mode of the cryo-grinder (the working temperature of the cryo-grinder is set to be minus 15 ℃) to cut nails into small blocks, putting the small blocks into a 2.0mL special EP pipe for grinding, adding three grinding steel balls, starting the cryo-grinder when the cryo-grinder descends to minus 15 ℃, and grinding the nails, wherein parameters are set as follows: operating frequency: 75Hz; run time: 60s; stop time: 15s; cycle times: 8, 8;
(3) Placing the EP pipe with the ground nails into a high-speed centrifuge, centrifuging for 3min at 12000r/min to centrifuge the nail scraps on the wall of the EP pipe to the bottom as much as possible, and removing the grinding steel balls;
(4) Adding 400 mu L of 5% Chelex-100 suspension, 10 mu L of 10mg/mL proteinase K solution and 10 mu L of 1.0mol/L DTT (dithiothreitol), shaking, mixing, centrifuging in a small-sized centrifuge for 10s, and throwing the liquid on the pipe wall to the bottom; placing the mixture in a thermostat at 56 ℃ for heat preservation, incubation and digestion overnight;
(5) Shaking and uniformly mixing the digested sample, putting the sample into a small-sized centrifuge for centrifugation for 10s, putting the sample into a thermostat at 100 ℃, and incubating for 8min again;
(6) Shaking and uniformly mixing the nail sample, and centrifuging in a high-speed centrifuge for 3min at 12000 r/min;
(7) Transferring the supernatant after centrifugation into another clean EP tube to obtain the free edge nuclear DNA of the nail.
As shown in FIG. 8, in the case of the accurate allele-typing control (Ladder and positive control genotyping site), the allele-typing peak height of the STR portion was not uniform, the peak was too low, and the allele-typing deletion occurred. The specific typing results are shown in Table 8.
TABLE 8 STR locus typing results
| Sample numbering | D3S1358 | vWA | D16S539 | CSF1PO | TPOX | D8S1179 | D21S11 | D18S51 |
| 2022-J221-F | 16,17 | 17,18 | 12 | / | 8,11 | 12,13 | / | / |
| Sample numbering | PentaE | D2S441 | D19S433 | TH01 | FGA | D22S1045 | D5S818 | D13S317 |
| 2022-J221-F | / | 11,14 | 13,14 | 9 | 22,24 | 16,17 | / | / |
| Sample numbering | D7S820 | D6S1043 | D10S1248 | D1S1656 | D12S391 | D2S1338 | PentaD | AMEL |
| 2022-J221-F | / | / | 13 | 15 | / | 19 | / | XY |
The results of fig. 8 and table 8 show that when the nail free edge sample is ground, the grinding temperature is high, which results in insufficient grinding and affects the effect of cell lysis, thereby resulting in disqualification of the nail free edge nuclear DNA typing pattern and failure of DNA extraction.
In summary, according to the method for extracting the free edge nuclear DNA of the nails, the free edge of the nails is ground into powder (the grinding environment temperature is low enough, the grinding frequency is high enough, the cycle times are high enough, the cell lysis effect is better), and then digestion is carried out, so that the success rate of extracting the free edge DNA of the nails can be greatly increased, the STR parting map of the extracted free edge nuclear DNA is qualified, and the available probability is greatly improved.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (6)
1. A method for extracting free edge nuclear DNA of a nail, comprising the steps of:
(1) Cutting the nails near the finger tips into small blocks, performing cold grinding into powder in a cold grinder pre-cooled to-70 ℃ to-60 ℃ in advance, and performing high-speed centrifugation; the parameters of the freeze-grinding include: operating frequency: 70Hz to 80Hz; run time: 50 s-70 s; stop time: 10 s-20 s; cycle times: 6 times to 10 times;
(2) Adding digestive juice to digest the nails; the digestive juice comprises: 3-7wt% of Chelex-100 suspension 350 [ mu ] L-450 [ mu ] L, 8 mg/mL-12 mg/mL of proteinase K solution 8 [ mu ] L-12 [ mu ] L and 0.8 mol/L-0.8 mol/L
1.2mol/L dithiothreitol 8 [ mu ] L-12 [ mu ] L; the digestion comprises: primary digestion is carried out for 6 to 24 hours at the temperature of 54 to 58 ℃ and then secondary digestion is carried out for 6 to 10 minutes at the temperature of 100 ℃;
(3) Centrifuging at high speed, and collecting supernatant.
2. The method for extracting free edge nuclear DNA of nails according to claim 1, wherein the cryogrinder is pre-cooled to-66℃to-64℃in advance in step (1).
3. The method of extracting free edge nuclear DNA of the nail of claim 1, wherein the parameters of the freeze-milling include: operating frequency: 74 Hz-76 Hz; run time: 55s-65 s; stop time: 14 s-16 s; cycle times: 7 times to 9 times.
4. The method for extracting free edge nuclear DNA of nails according to claim 1, wherein after adding digestive juice in the step (2), shaking, mixing and centrifuging for 8-12 s, and then performing primary digestion; and/or after primary digestion, shaking, uniformly mixing and centrifuging for 8-12 s, and then performing secondary digestion.
5. The method for extracting free edge nuclear DNA of nails according to any one of claims 1 to 4, wherein in the step (1), the surface of the nails is scraped clean by a surgical blade before the nails are cut into small pieces;
and/or, the high-speed centrifugation in the step (1) is performed at a speed of 10000 r/min-14000 r/min for 2 min-4 min;
and/or, the high-speed centrifugation in the step (3) is performed at a speed of 10000 r/min-14000 r/min for 2 min-4 min.
6. Use of the method for extracting free edge nuclear DNA of nails according to any one of claims 1 to 5 in forensic physical evidence testing.
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| CN110656106A (en) * | 2018-06-29 | 2020-01-07 | 重庆市公安局巴南区分局 | Method for extracting free DNA from somatotype biological sample |
| CN111154751A (en) * | 2020-01-14 | 2020-05-15 | 东北林业大学 | Method for efficiently extracting DNA (deoxyribonucleic acid) in hair shaft |
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| CN110656106A (en) * | 2018-06-29 | 2020-01-07 | 重庆市公安局巴南区分局 | Method for extracting free DNA from somatotype biological sample |
| CN111154751A (en) * | 2020-01-14 | 2020-05-15 | 东北林业大学 | Method for efficiently extracting DNA (deoxyribonucleic acid) in hair shaft |
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