CN114736898A - Method for extracting DNA of nail free edge nucleus and application - Google Patents
Method for extracting DNA of nail free edge nucleus and application Download PDFInfo
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Abstract
The invention discloses a method for extracting DNA of a free edge nucleus of a nail and application thereof, wherein the nail close to the tip of the nail is cut into small pieces, and is ground into powder in a cryogrinding instrument precooled to-70 ℃ to-60 ℃ in advance, and then is centrifuged at high speed; adding digestive juice to digest the nail; centrifuging at high speed, and collecting supernatant. The method for extracting the DNA of the free edge nuclei of the nails can greatly increase the success rate of extracting the DNA of the free edge nuclei of the nails, and the STR typing map of the extracted DNA of the free edge nuclei is qualified, so that the available probability is greatly improved, and the re-extraction rate and the probability of repeated sampling are reduced. The whole extraction process is simplified, the harm to the body in the use process of the organic reagent is reduced, and the cost is low.
Description
Technical Field
The invention belongs to the field of biotechnology, and particularly relates to a method for extracting nuclear DNA from a nail free edge 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 identification of biological test materials related to human bodies in cases, and proves the case fact by the components and characteristics of the biological test materials, wherein the biological test materials usually comprise blood, semen, vaginal secretion, saliva, amniotic fluid and spots thereof; hair, nails, bone, and teeth; various tissues, organs and fragments thereof, etc. of human body. The main task of forensic physical evidence inspection is to solve the problems of personal identification and paternity identification in judicial practice.
With the increasing depth of DNA genetic markers, DNA typing has become the main means of forensic physical evidence testing. Individual identification is performed by DNA typing rather than by sequencing the entire genome. DNA typing also has the advantage of obtaining the same result from any body fluid or tissue containing cells, and thus, in the actual identification work, the selection of the test material is superior or inferior. The fingernails are often the preferred materials for the identified persons because of their non-invasive, covert, and easily accessible characteristics. The fingernails are easy to store and transport, and can be selected as identification materials for patients with serious diseases or patients with a history of blood transfusion. The fingernails have strong corrosion resistance, so the fingernails can also be selected as the inspection materials of corpses.
The nail is formed by keratinization of epidermal cells, consists of a plurality of layers of firmly connected keratinocytes, and is rich in keratin in cells, including three parts, namely a free edge, a nail body and a nail root. In the case inspection process of some criminal cases, the nail DNA inspection needs to pull out the whole nail and digest the nail body or the nail root part of the nail to obtain a satisfactory DNA inspection result. However, in the field of paternity testing, it is not practical to pick up the entire nail for testing, and generally, only the free edge of the nail can be obtained for testing. The lower part of the nail free edge is not supported, and the nail cells lack the nutrition supply of the nail bed, so compared with other parts of the nail, the cell keratinization degree is higher, the cell nucleus is degenerated, the nuclear DNA is more seriously degraded, the content is very low, the DNA extraction difficulty is very high, and simultaneously, the extraction difficulty of the DNA is increased because the nail free edge has a compact structure.
At present, an organic method combined with a Chelex-100 extraction method, a Chelex-100 combined adsorption column method or a magnetic bead method is generally selected for extracting the nail DNA, and the pretreatment operation steps of the methods are various: generally, a proper amount of fingernails are put into a high-speed centrifuge tube, 1.5mL of 10% SDS is added, the mixture is shaken and soaked for about 30min, deionized water is repeatedly soaked and washed for 2-3 times, then double distilled water is used for rinsing for 2-3 times, and after drying, the mixture is cut into small pieces by ophthalmic scissors. The series of operations are long in time consumption, easy to cause pollution, difficult to extract enough amount of DNA, poor in experimental amplification effect and difficult to electrophorese to obtain a complete map. And the organic solvent causes a certain degree of harm to the body. DNA extraction kits for nail free edge are also available on the market, but are expensive and high in cost.
Therefore, because of the special structure of the free edge of the nail, the extraction of DNA is difficult, and the DNA is not generally used as a preferable identification material for forensic physical evidence at present. The method for extracting the DNA is simple, accurate and low in cost, and has great significance.
Disclosure of Invention
Based on this, one of the objectives of the present invention is to provide a method for extracting DNA of nail free edge nuclei, which can greatly improve the success rate and quality of extracting DNA of free edge nuclei, and is simple, fast and needs no organic solvent.
The specific technical scheme for realizing the aim of the invention comprises the following steps:
a method for extracting DNA of a nail free edge nucleus comprises the following steps:
(1) cutting the nails close to the finger tips into small pieces, carrying out freeze grinding in a freeze grinding instrument pre-cooled to-70 ℃ to-60 ℃ in advance to obtain powder, and centrifuging at a high speed;
(2) adding digestive juice to digest the nail;
(3) and centrifuging at high speed, and taking the supernatant to obtain the product.
In some of the embodiments, the cryo-mill in step (1) is pre-cooled to-66 ℃ to-64 ℃ in advance.
In some of these embodiments, the parameters of the cryomilling in step (1) include: operating frequency: 70Hz to 80 Hz; operating time: 50 s-70 s; stopping time: 10 s-20 s; cycle number: 6 times to 10 times.
In some of these embodiments, the parameters of the cryomilling include: operating frequency: 74Hz to 76 Hz; operating time: 55 s-65 s; stopping time: 14s to 16 s; cycle number: 7 times to 9 times.
In some of these embodiments, the digesting in step (2) comprises: primary digestion is carried out for 6 to 24 hours at 54 to 58 ℃, and then 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 digestion is carried out again at 100 ℃ for 8 min.
In some embodiments, after the digestive juice is added in the step (2), shaking, uniformly mixing, centrifuging for 8-12 s, and then performing primary digestion; and/or after primary digestion, shaking, uniformly mixing, centrifuging for 8-12 s, and then carrying out secondary digestion.
In some embodiments, the digestive juice in step (3) comprises: 350-450 muL of 3-7 wt% Chelex-100 suspension, 8-12 muL of 8-12 mg/mL proteinase K solution and 8-12 muL of 0.8-1.2 mol/L dithiothreitol.
In some embodiments, in step (1), the nail surface is cleaned by scraping with a surgical blade before cutting the nail into small pieces.
In some embodiments, the high-speed centrifugation in the step (1) is carried out at 10000 r/min-14000 r/min for 2 min-4 min;
in some embodiments, the high speed centrifugation in step (3) is performed at 10000 r/min-14000 r/min for 2 min-4 min.
The invention also provides the DNA of the nail free edge nucleus extracted by the method.
The invention also provides application of the above DNA in forensic physical evidence inspection.
Compared with the prior art, the invention has the following beneficial effects:
the method for extracting the DNA of the nail free edge nuclear greatly increases the success rate of extracting the DNA of the nail free edge nuclear by freezing and grinding the nail free edge under the freezing condition, accurately controls the temperature and the parameters of the freezing and grinding and then digests the powder after the freezing and grinding, greatly improves the probability of the STR typing map being qualified and available and reduces the probability of re-extraction rate and repeated sampling. In addition, the whole extraction process is simplified, a plurality of complex pretreatment steps are omitted, the use of organic solvents is avoided, the harm to human bodies in the use process of organic reagents is reduced, and meanwhile, the cost is low.
Drawings
FIG. 1 is a STR part locus map of the DNA of the nail free edge nucleus extracted in example 1 of the present invention.
FIG. 2 is a STR partial locus map of the DNA of the nail free edge nucleus extracted in example 2 of the present invention.
FIG. 3 is a STR partial locus map of the DNA of the nail free edge nucleus extracted in example 3 of the present invention.
FIG. 4 is a STR partial locus map of the DNA of the nail free edge nucleus extracted in comparative example 1 of the present invention.
FIG. 5 is a STR partial locus map of the DNA of the nail free edge nucleus extracted in comparative example 2 of the present invention.
FIG. 6 is a STR partial locus map of the DNA of the nail free edge nucleus extracted in comparative example 3 of the present invention.
FIG. 7 is a STR partial locus map of the DNA of the nail free edge nucleus extracted in comparative example 4 of the present invention.
FIG. 8 is a STR partial locus map of the DNA of the nail free edge nucleus extracted in comparative example 5 of the present invention.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the following description. The present 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. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
In one aspect of the present invention, a method for extracting DNA from a DNA of a nail free edge is provided, which comprises the following steps:
(1) selecting 3-10 mg of nails which are close to the tips of the fingers as much as possible, and scraping the surfaces of the nails by using a surgical blade to prevent the carried gene pollution and the inhibition effect of non-gene impurities;
(2) cutting the nails into small pieces, putting the small pieces into a special grinding EP tube, and grinding the nails into powder by using a freeze grinding instrument (note that before the experiment begins, a refrigeration mode of the freeze grinding instrument is started to reduce the grinding environment temperature to-70-60 ℃), wherein the parameters are set as follows: operating frequency: 70Hz to 80 Hz; operating time: 50 s-70 s; stopping time: 10 s-20 s; cycle number: 6-10 times (the operation parameters can be determined according to actual conditions, and when the grinding effect is not good, the cycle number can be increased). Preferably, the parameters of the cryomill are set as: operating frequency: 75 Hz; operating time: 60S; stopping time: 15S; cycle number: 8. the nail is used as keratinized hard tissue, the tissue is compact, the brittleness of the nail can be increased by freezing, grinding is more sufficient, the nail can be directly frozen to a cell layer after grinding and crushing, keratinized cell membranes are sufficiently destroyed and depolymerized, and cell lysis and digestion are more thorough.
(3) Putting the EP tube filled with the nail powder into a high-speed centrifuge, centrifuging for 2-4 min at 10000-14000 r/min to ensure that the nail powder on the wall of the EP tube is centrifuged to the bottom as much as possible; preferably, the rotating speed of the high-speed centrifuge is 12000r/min, and the centrifugation time is 3 min;
(4) adding 350-450 mu L of Chelex-100 suspension with the mass concentration of 3-7%, 8-12 mu L of 8-12 mg/mL proteinase K liquid and 8-12 mu L of 0.8-1.2 mol/L DTT (dithiothreitol), vibrating and mixing uniformly, putting into a small centrifuge for centrifuging for 8-12 s, and throwing the liquid on the tube wall to the bottom; putting the mixture into a thermostat with the temperature of 54-58 ℃ for heat preservation, incubation and digestion for more than 6 hours (preferably incubating overnight, and the effect is better);
(5) after shaking and mixing uniformly, putting the mixture into a small centrifuge for centrifuging for 8-12 s, putting the centrifuge into a thermostat with the temperature of 100 ℃, and incubating for 6-10 min again;
(6) and after shaking and uniform mixing, centrifuging the mixture in a high-speed centrifuge at 10000 r/min-14000 r/min for 2 min-4 min (preferably, the rotating speed of the high-speed centrifuge is 12000r/min, and the centrifuging time is 3min), and taking supernatant to obtain the finished product.
The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.
Example 1A method for extracting DNA from the DNA of the nail free edge
The method for extracting DNA of the nail free edge nucleus comprises the following steps:
(1) selecting about 7mg of sample (sample No. 2022-J18-Z) of the nail free edge of the No. 1 volunteer close to the tip of the finger, and scraping the surface of the nail with a surgical blade;
(2) starting a refrigeration mode of the freeze grinding instrument, cutting the nail into small pieces, putting the small pieces into a special 2.0mL grinding EP pipe, adding three grinding steel balls, starting the freeze grinding instrument when the freeze grinding instrument is lowered to-65 ℃, grinding the nail into powder, and setting parameters as follows: operating frequency: 75 Hz; operating time: 60 s; stopping time: 15 s; cycle number: 8;
(3) putting the EP tube filled with the nail powder into a high-speed centrifuge, centrifuging at 12000r/min for 3min to ensure that the nail powder on the wall of the EP tube is centrifuged to the bottom as much as possible, and removing the grinding steel balls;
(4) adding 400 mu L of Chelex-100 suspension with the mass concentration of 5%, 10 mu L of 10mg/mL proteinase K liquid and 10 mu L of 1.0mol/L DTT (dithiothreitol), shaking and uniformly mixing, putting into a small centrifuge for centrifugation for 10s, and throwing the liquid on the tube wall to the bottom; putting the mixture into a thermostat with the temperature of 56 ℃ for heat preservation, incubation and digestion overnight;
(5) after the digested sample is shaken and uniformly mixed, the mixture is placed into a small centrifuge for centrifugation for 10s, and then placed into a thermostat with the temperature of 100 ℃ for incubation for 8min again;
(6) after the nail samples are shaken and uniformly mixed, the mixture is centrifuged for 3min at 12000r/min in a high-speed centrifuge;
(7) and transferring the centrifuged supernatant into another clean EP tube, namely the DNA of the nail free edge nucleus.
Washington's chart using a fluorescent marker from 1. mu.L of the DNA extracted in this exampleTMPerforming multiplex STR locus amplification by using the platinum PCR amplification kit (by using a method in a kit instruction); the amplified product was electrophoresed using an American ABI 3500XL genetic analyzer and used
And D, performing data analysis by using ID-X software to derive an STR map.
The STR partial locus map is shown in figure 1, under the condition that an allele typing contrast (Ladder and positive contrast genotyping site) is accurate, the peak height of each allele typing of the map reaches over 100bp, and most of the alleles are more balanced in typing. 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- |
15,16 | 14,19 | 9,11 | 12 | 8 | 13,15 | 29 | 15 |
| Sample numbering | PentaE | D2S441 | D19S433 | TH01 | FGA | D22S1045 | D5S818 | D13S317 |
| 2022-J18- |
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- |
11,12 | 11 | 16 | 11,16 | 17,19 | 20,22 | 9,12 | X,Y |
The results in FIG. 1 and Table 1 show that the method of this example successfully extracted DNA from the nail free edge nuclei and that the map quality was good.
Example 2A method for extracting DNA from the edge of the nail free nucleus
The method for extracting DNA of the nail free edge nucleus comprises the following steps:
(1) selecting a sample (sample number: 2021-J191-Z2) of the free edge of the fingernail of the No. 2 volunteer close to the tip of the fingernail, and scraping the surface of the fingernail clean by a surgical blade;
(2) the refrigeration mode of the freeze grinder is started firstly, the nail is cut into small pieces, 2.0mL of special grinding EP pipe is put in, three grinding steel balls are added, when the freeze grinder is lowered to-65 ℃, the freeze grinder is started, the nail is ground into powder, and the parameters are set as follows: operating frequency: 75 Hz; operating time: 60 s; stopping time: 15 s; cycle number: 7;
(3) putting the EP tube filled with the nail powder into a high-speed centrifuge, centrifuging at 12000r/min for 3min to ensure that the nail powder on the wall of the EP tube is centrifuged to the bottom as much as possible, and removing the grinding steel balls;
(4) adding 400 mu L of Chelex-100 suspension with the mass concentration of 5%, 10 mu L of 10mg/mL proteinase K liquid and 10 mu L of 1.0mol/L DTT (dithiothreitol), shaking and uniformly mixing, putting into a small centrifuge for centrifugation for 10s, and throwing the liquid on the tube wall to the bottom; putting the mixture into a thermostat with the temperature of 56 ℃, preserving heat, incubating and digesting the mixture overnight;
(5) after the digested sample is shaken and uniformly mixed, the mixture is placed into a small centrifuge for centrifugation for 10s, and then placed into a thermostat with the temperature of 100 ℃ for incubation for 8min again;
(6) after the nail samples are shaken and uniformly mixed, the mixture is centrifuged for 3min at 12000r/min in a high-speed centrifuge;
(7) and transferring the centrifuged supernatant into another clean EP tube, namely the DNA of the nail free edge nucleus.
The STR partial locus map is shown in figure 2, under the condition that an allele typing contrast (Ladder and positive contrast genotyping site) is accurate, the peak height of each allele typing of the map reaches over 100bp, and most of the alleles are more balanced in typing. 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- |
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- |
11 | 14,19 | 14 | 16 | 18,20 | 20,25 | 9,12 | X,Y |
The results in FIG. 2 and Table 2 show that the method of this example successfully extracts DNA from the nail free edge nuclei and that the map quality is good.
Example 3A method for extracting DNA from the DNA of the nail-free edge nucleus
The method for extracting DNA of the nail free edge nucleus comprises the following steps:
(1) selecting 10mg of sample (sample number: 2021-J216-F) of the nail free edge of No. 3 volunteer near the tip of the finger, and scraping the surface of the nail with a surgical blade;
(2) the refrigeration mode of the freeze grinder is started firstly, the nail is cut into small pieces, 2.0mL of special grinding EP pipe is put in, three grinding steel balls are added, when the freeze grinder is lowered to-65 ℃, the freeze grinder is started, the nail is ground into powder, and the parameters are set as follows: operating frequency: 75 Hz; operating time: 60 s; stopping time: 15 s; cycle number: 6;
(3) putting the EP tube filled with the nail powder into a high-speed centrifuge, centrifuging at 12000r/min for 3min to ensure that the nail powder on the wall of the EP tube is centrifuged to the bottom as much as possible, and removing the grinding steel balls;
(4) adding 400 mu L of Chelex-100 suspension with the mass concentration of 5%, 10 mu L of 10mg/mL proteinase K liquid and 10 mu L of 1.0mol/L DTT (dithiothreitol), shaking and uniformly mixing, putting into a small centrifuge for centrifugation for 10s, and throwing the liquid on the tube wall to the bottom; putting the mixture into a thermostat with the temperature of 56 ℃, preserving heat, incubating and digesting the mixture overnight;
(5) after the digested sample is shaken and uniformly mixed, the mixture is placed into a small centrifuge for centrifugation for 10s, and then placed into a thermostat with the temperature of 100 ℃ for incubation for 8min again;
(6) vibrating and uniformly mixing the nail samples, and centrifuging for 3min at 12000r/min in a high-speed centrifuge;
(7) and transferring the centrifuged supernatant into another clean EP tube to obtain the DNA of the nail free edge nucleus.
The STR partial locus map is shown in figure 3, under the condition that an allele typing contrast (Ladder and positive contrast genotyping site) is accurate, the peak height of each allele typing of the map reaches over 100bp, and most of the alleles are more balanced in typing. Specific typing results are shown in table 3.
TABLE 3 STR locus typing results
The results in FIG. 3 and Table 3 show that the method of this example successfully extracts DNA from the nail free edge nuclei, and the map is qualified and of good quality.
Comparative example 1A method for extracting DNA from the edge of the nail free nucleus
The method for extracting the DNA of the nail free edge nucleus comprises the following steps:
(1) selecting about 7mg of sample (sample No. 2022-J18-Z) of the nail free edge of the No. 1 volunteer close to the tip of the finger, and scraping the surface of the nail with a surgical blade;
(2) cutting nails into small pieces, putting the small pieces 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 uniformly mixing, putting the small centrifuge into the small centrifuge for centrifuging for 10s, and throwing the liquid on the tube wall to the bottom; putting the mixture into a thermostat with the temperature of 56 ℃, preserving heat, incubating and digesting the mixture overnight;
(3) after the digested sample is shaken and uniformly mixed, the mixture is placed into a small centrifugal machine for centrifugation for 10s, and then placed into a 100 ℃ thermostat for incubation for 8min again;
(4) after the nail samples are shaken and uniformly mixed, the mixture is centrifuged for 3min at 12000r/min in a high-speed centrifuge;
(5) and transferring the centrifuged supernatant into another clean EP tube to obtain the DNA.
The STR partial locus map is shown in FIG. 4, and under the condition that the allelic typing controls (Ladder and positive control genotyping sites) are accurate, the peak heights of partial allelic typing of the map are not balanced, the peak values are too low, and allelic typing deletion occurs. Specific typing results are shown in table 4.
TABLE 4 STR locus typing results
Note: in the table, "/" indicates site deletion
The results in FIG. 4 and Table 4 show that when the DNA typing map of the formazan free edge nuclei is not qualified and the DNA extraction fails, the effect of cell lysis is affected when the formazan free edge samples are directly digested without being frozen and ground.
Comparative example 2A method for extracting DNA from the edge of the nail free nucleus
The method for extracting the DNA of the nail free edge nucleus comprises the following steps:
(1) selecting 10mg of sample (sample number: 2021-J137-F) of the nail free edge close to the tip of the finger of No. 6 volunteer, and scraping the surface of the nail with a surgical blade;
(2) the refrigeration mode of the freeze grinder is started firstly, the nail is cut into small pieces, 2.0mL of special grinding EP pipe is put in, three grinding steel balls are added, when the freeze grinder is lowered to-65 ℃, the freeze grinder is started, the nail is ground into powder, and the parameters are set as follows: operating frequency: 35 Hz; operating time: 60 s; stopping time: 15 s; cycle number: 2;
(3) putting the EP tube filled with the nail powder into a high-speed centrifuge, centrifuging at 12000r/min for 3min to ensure that the nail powder on the wall of the EP tube is centrifuged to the bottom as much as possible, and removing the grinding steel balls;
(4) adding 400 mu L of Chelex-100 suspension with the mass concentration of 5%, 10 mu L of 10mg/mL proteinase K liquid and 10 mu L of 1.0mol/L DTT (dithiothreitol), shaking and uniformly mixing, putting into a small centrifuge for centrifugation for 10s, and throwing the liquid on the tube wall to the bottom; putting the mixture into a thermostat with the temperature of 56 ℃, preserving heat, incubating and digesting the mixture overnight;
(5) after the digested sample is shaken and uniformly mixed, the mixture is placed into a small centrifugal machine for centrifugation for 10s, and then placed into a 100 ℃ thermostat for incubation for 8min again;
(6) after the nail samples are shaken and uniformly mixed, the mixture is centrifuged for 3min at 12000r/min in a high-speed centrifuge;
(7) and transferring the centrifuged supernatant into another clean EP tube, namely the DNA of the nail free edge nucleus.
The STR partial locus map is shown in FIG. 5, and in the case that the allelic typing controls (Ladder and positive control genotyping site) are accurate, the heights of the allelic typing peaks of the partial locus map are not balanced, the peak values are too low, and allelic typing deletion occurs. 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- |
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: in the table, "/" indicates site deletion
The results in fig. 5 and table 5 show that when freeze-grinding is performed on the nail free edge sample, the grinding frequency is low, and the grinding time is short, so that the grinding is insufficient, the nail free edge nuclear DNA typing map is unqualified, and the DNA extraction fails.
Comparative example 3A method for extracting DNA from the edge of the nail free nucleus
The method for extracting the DNA of the nail free edge nucleus comprises the following steps:
(1) selecting about 7mg of a sample (sample number: 2021-J179-Z) of the nail free edge of No. 4 volunteers close to the tip of the finger, and scraping the surface of the nail with a surgical blade;
(2) cutting the fingernails into small pieces, grinding the small pieces in a grinding bowl for about 60 minutes, and putting the ground nail pieces into a 2.0mLEP tube;
(3) adding 400 mu L of Chelex-100 suspension with the mass concentration of 5%, 10 mu L of 10mg/mL proteinase K liquid and 10 mu L of 1.0mol/L DTT (dithiothreitol), shaking and uniformly mixing, putting into a small centrifuge for centrifugation for 10s, and throwing the liquid on the tube wall to the bottom; putting the mixture into a thermostat with the temperature of 56 ℃, preserving heat, incubating and digesting the mixture overnight;
(4) after the digested sample is shaken and uniformly mixed, the mixture is placed into a small centrifugal machine for centrifugation for 10s, and then placed into a 100 ℃ thermostat for incubation for 8min again;
(5) after the nail samples are shaken and uniformly mixed, the mixture is centrifuged for 3min at 12000r/min in a high-speed centrifuge;
(6) and transferring the centrifuged supernatant into another clean EP tube to obtain the DNA of the nail free edge nucleus.
The STR partial locus map is shown in FIG. 6, and under the condition that the allelic typing controls (Ladder and positive control genotyping sites) are accurate, the peak heights of partial allelic typing of the map are not balanced, the peak values are too low, and allelic typing deletion occurs. 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- |
15 | 14 | 9 | 11 | 8,9 | 13,15 | / | / |
| Sample numbering | PentaE | D2S441 | D19S433 | TH01 | FGA | D22S1045 | D5S818 | D13S317 |
| 2021-J179- |
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- |
11 | / | 13,16 | / | 19,20 | 18,20 | / | X,Y |
Note: in the table, "/" indicates site deletion
The results in fig. 6 and table 6 show that when the formazan free edge samples are not frozen, but ground by using the grinding bowl directly and then digested, the cell lysis effect is affected, and the formazan free edge nuclear DNA typing map is not qualified, and the DNA extraction fails.
Comparative example 4A method for extracting DNA from the edge of the nail free nucleus
The method for extracting DNA of the nail free edge nucleus in the comparative example adopts the existing DNA extraction method, and specifically comprises the following steps:
(1) selecting about 7mg of a fingernail free edge sample (sample number: 2022-J118-Z2) of No. 5 volunteer close to the end of a finger, putting the fingernail into a 2.0mLEP tube, adding 1.5mL of 10% SDS, placing on a MicroBio shaking apparatus, shaking and soaking for 30min at the maximum speed, repeatedly soaking and washing for 2-3 times by using deionized water, rinsing for 2-3 times by using double distilled water, cutting the fingernail into small pieces after drying, and putting into the 2.0mLEP tube.
(2) Adding STE buffer solution 400ul, protease K solution 40 uL of 10mg/mL, DTT40 uL of 1.0mol/L and SDS of 10% 100 uL, shaking and mixing uniformly, putting the mixture into a small centrifuge for centrifugation for 10s, throwing the liquid on the tube wall to the bottom, putting the tube wall into a thermostat of 56 ℃ for heat preservation, hatching and digesting overnight.
(3) The liquid was transferred to an EP tube containing an equal volume of an organic solvent (saturated phenol: chloroform: isoamyl alcohol: 25: 24: 1), mixed and shaken, and centrifuged at 13,000rpm for 3 min.
(4) Transferring the supernatant into another clean centrifuge tube, adding 2.5 times of cold absolute ethyl alcohol, freezing and storing for 10min, centrifuging at 13,000rpm for 5min, discarding the supernatant, and air-drying the residual ethyl alcohol at room temperature;
(5) adding 200 mu L of 5% Chelex-100 suspension into the air-dried DNA, putting the DNA into a thermostat with the temperature of 56 ℃ for digestion for 6 hours, taking out the DNA, violently shaking the DNA for 5-10 seconds at 13,000rpm, and centrifuging the DNA for 3 min;
(6) and transferring the centrifuged supernatant into another clean EP tube to obtain the DNA of the nail free edge nucleus.
The STR partial locus map is shown in figure 7, under the condition that an allelic typing control (Ladder and a positive control genotyping site) is accurate, the peak height of each allelic typing of the map reaches over 100bp, and most of allelic typing is balanced. 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- |
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- |
10,16 | 11,14 | 14,14.2 | 9 | 23 | 15 | 11 | 11 |
| Sample numbering | D7S820 | D6S1043 | D10S1248 | D1S1656 | D12S391 | D2S1338 | PentaD | AMEL |
| 2022-J118- |
11,12 | 12,19 | 13,15 | 11,12 | 22 | 17,24 | 8,11 | X,Y |
The results in FIG. 7 and Table 7 show that the spectrum is qualified and usable when the method of the comparative example (organic method combined with Chelex-100 extraction method) is used for successfully extracting the DNA of the finger nail free edge nucleus, but the DNA extraction process is more complicated and takes longer time.
Comparative example 5A method for extracting DNA of nail free edge nucleus
The method for extracting the DNA of the nail free edge nucleus comprises the following steps:
(1) selecting about 8mg of sample (sample number: 2021-J221-F) of No. 7 fingernail free edge close to the tip of the finger of the volunteer, and scraping the surface of the fingernail with a surgical blade;
(2) firstly, starting a refrigeration mode of the freeze grinder (the working temperature of the freeze grinder is set to-15 ℃) to cut the fingernails into small pieces, putting the small pieces into a 2.0mL special EP grinding pipe, adding three grinding steel balls, starting the freeze grinder when the freeze grinder is lowered to-15 ℃, grinding the fingernails, and setting parameters as follows: operating frequency: 75 Hz; operating time: 60 s; stopping time: 15 s; cycle number: 8;
(3) putting the EP tube with the grinded fingernails into a high-speed centrifuge, centrifuging at 12000r/min for 3min to ensure that the fingernail powder on the wall of the EP tube is centrifuged to the bottom as much as possible, and removing the grinded steel balls;
(4) adding 400 mu L of Chelex-100 suspension with the mass concentration of 5%, 10 mu L of 10mg/mL proteinase K liquid and 10 mu L of 1.0mol/L DTT (dithiothreitol), shaking and uniformly mixing, putting into a small centrifuge for centrifugation for 10s, and throwing the liquid on the tube wall to the bottom; putting the mixture into a thermostat with the temperature of 56 ℃, preserving heat, incubating and digesting the mixture overnight;
(5) after the digested sample is shaken and uniformly mixed, the mixture is placed into a small centrifugal machine for centrifugation for 10s, and then placed into a 100 ℃ thermostat for incubation for 8min again;
(6) after the nail samples are shaken and uniformly mixed, the mixture is centrifuged for 3min at 12000r/min in a high-speed centrifuge;
(7) and transferring the centrifuged supernatant into another clean EP tube, namely the DNA of the nail free edge nucleus.
The STR partial locus map is shown in FIG. 8, and under the condition that the allelic typing controls (Ladder and positive control genotyping sites) are accurate, the peak heights of partial allelic typing of the map are not balanced, the peak values are too low, and allelic typing deletion occurs. 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- |
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 in FIG. 8 and Table 8 show that when grinding the samples on the nail free edge, the grinding temperature is higher, which leads to insufficient grinding and influences the cell lysis effect, thus leading to disqualification of the typing map of the nail free edge nuclear DNA and failure of DNA extraction.
In conclusion, the method for extracting the DNA of the nail free edge nuclei of the invention can greatly increase the success rate of extracting the DNA of the nail free edge nuclei by grinding the nail free edge into powder (the grinding environment temperature needs to be low enough, the grinding frequency needs to be high enough, and the cycle times need to be sufficient enough to ensure that the cell lysis effect is better), and then digesting the powder, and the STR typing map of the extracted DNA of the nail free edge nuclei is qualified, so that the available probability is greatly improved.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the present patent should be subject to the appended claims.
Claims (10)
1. A method for extracting DNA of a nail free edge nucleus, which is characterized by comprising the following steps:
(1) cutting the nails close to the finger tips into small pieces, carrying out freeze grinding in a freeze grinding instrument pre-cooled to-70 ℃ to-60 ℃ in advance to obtain powder, and centrifuging at a high speed;
(2) adding digestive juice to digest the nail;
(3) and centrifuging at high speed, and taking the supernatant to obtain the product.
2. The method for extracting DNA from nail-free edge nuclei according to claim 1, wherein the cryo-grinder in step (1) is pre-cooled to-66 ℃ to-64 ℃ in advance.
3. The method for extracting DNA from nail free edge nuclei according to claim 1, wherein the parameters of the freeze-grinding in the step (1) include: operating frequency: 70Hz to 80 Hz; operating time: 50 s-70 s; stopping time: 10 s-20 s; cycle number: 6 times to 10 times.
4. The method for extracting DNA from nail free edge nuclei of claim 3, wherein the parameters of the freeze-grinding include: operating frequency: 74Hz to 76 Hz; operating time: 55 s-65 s; stopping time: 14s to 16 s; cycle number: 7 times to 9 times.
5. The method for extracting DNA from the nail-free edge nuclei according to any one of claims 1 to 4, wherein the digestion in the step (2) comprises: primary digestion is carried out for 6 to 24 hours at 54 to 58 ℃, and then secondary digestion is carried out for 6 to 10 minutes at 100 ℃.
6. The method for extracting the DNA of the nail free edge nucleus according to claim 5, wherein after adding the digestive juice, shaking, uniformly mixing and centrifuging for 8-12 s, and then carrying out primary digestion; and/or after primary digestion, shaking, uniformly mixing, centrifuging for 8-12 s, and then carrying out secondary digestion.
7. The method for extracting DNA from the nail free edge nucleus according to any one of claims 1 to 4, wherein the digestion solution in the step (3) comprises: 350-450 muL of 3-7 wt% Chelex-100 suspension, 8-12 muL of 8-12 mg/mL proteinase K solution and 8-12 muL of 0.8-1.2 mol/L dithiothreitol.
8. The method for extracting DNA from the free edge of the nail according to any one of claims 1 to 4, wherein in the step (1), the surface of the nail is scraped clean by a surgical blade before the nail is cut into small pieces;
and/or, the high-speed centrifugation speed in the step (1) is 10000 r/min-14000 r/min, and the time is 2 min-4 min;
and/or the high-speed centrifugation speed in the step (3) is 10000 r/min-14000 r/min, and the time is 2 min-4 min.
9. The method according to any one of claims 1 to 8, wherein the DNA is isolated from the nail free edge nuclei.
10. Use of the nail margin nuclear DNA of claim 9 in forensic physical evidence testing.
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