CN102229927B - Improve method and the reagent of rate of extraction of DNAs of castoff cells in case trace sample - Google Patents
Improve method and the reagent of rate of extraction of DNAs of castoff cells in case trace sample Download PDFInfo
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Abstract
The present invention relates to a kind of method and the reagent that improve rate of extraction of DNAs of castoff cells in case trace sample.Case sample amount is micro-, the problem such as cell keratinization and 16 STR site multiplex PCR composite amplifications, makes cast-off cells DNA extraction be difficult point in medical jurisprudence always.Find the bath of protease digestion, 70-100 degree temperature after deliberation, the combination of PEG-ethanol magnetic bead articulated system 3 can high efficiency extraction minim DNA, obtain the needs that the DNA fragmentation that is of convenient length can meet 16 STR site composite amplifications in case detection, achieve the lifting that case is detected as power.Protease digestion, temperature bath make the efficient released dna of cast-off cells, and structure is relatively complete, is easily combined with magnetic bead in PEG-ethanol system.The use of PEG-ethanol articulated system not only promotes that magnetic bead is to the efficient adsorption of DNA, and ensure that DNA structure is relatively complete, obtains the needs that the DNA fragmentation that is of convenient length can meet 16 STR site composite amplifications in case detection.
Description
Technical field
The present invention relates to method and the reagent of case trace sample cast-off cells DNA high efficiency extraction, belong to forensic DNA detection field.
Background technology
Since round pcr is applied to the inspection of biological evidence, after especially application str locus seat is analyzed, DNA extraction just becomes the key link of forensic dna polymorphism analysis, and the quality of DNA sample quality will be directly connected to the success or failure of subsequent experimental.But in actual inspection case, increasing case is due to the effect of the factor such as counterreconnaissance consciousness enhancing of case nature, commit a crime course and offender, in some site inspections, be difficult to find the standard biologic samples such as blood cake, seminal stain, hair, cigarette end, the sample that therefore may be loaded with human body case trace sample just becomes the important material evidence of cracking of cases.
Human body case trace sample amount is micro-, and mostly is keratinization, and nucleus is many degenerates, is attached on the various carriers with human contact.Its carrier has following features: 1. the sample big area such as micro-epidermic cell is distributed in carrier surface and is combined closely with carrier, dirt, is difficult to collect transfer; 2. the shelf time is long or improper, and DNA easily degrades and is difficult to satisfied 16 STR site multiplex PCR composite amplifications (interior 16 templates of pipe increase simultaneously); 3. this type of sample carrier is many through washing, containing PCR inhibition.Therefore, the extraction difficulty of case trace sample DNA is large, lacks stable extracting method, easily causes the failure of inspection case.
At present, according to the case trace amount of sample and the difference of its place carrier, the method for DNA extraction is also different.The sample that the human contact faces such as clothing, footwear, socks case trace sample that is comparatively large, that may contain is more, after many case trace samples by case trace sample absorption apparatus collection sample, extract by Automation workstation or chelex-100 method, by extending incubation period, increasing the amount of Proteinase K, reducing the methods such as elution volume, to reach the object improving DNA profiling concentration.And when running into the wooden handle of various instrument, as the fingerprint on the wooden handle of axe, all kinds of carrier, when as less in human contact positions such as the finger marks on bowl cover, carrier is to the sample that case trace sample adsorptivity is stronger, reviewer is generally the method taking different extractions according to the extraction experience of oneself, but is all often the cross-reference of plurality of reagents box and the mixing application of multiple extracting method.As hatched with Chelex-100 and Proteinase K after several hours through Microcon-100 purification column purified concentration DNA; Or extract DNA profiling with other test kits as QIAGENM48 magnetic bead kit, what have also uses chloroform purification step in organic method wherein.The DNA amount that these methods are extracted less, purity is low, length is imperfect, not only uses expensive import reagent, and complex steps, longer, complicated operation consuming time, personal experience's impact are comparatively large, and success ratio is lower.
The present inventor optimizes cracking, articulated system invention improves case trace sample DNA extraction efficiency method and reagent, solve the above problem run in DNA extraction process, and be successfully applied to actual inspection case.
Summary of the invention
The technical problem to be solved in the present invention is, system optimization cracking, combination, cleaning system solve the extraction difficult problem only containing minim DNA in case sample, realize easy, quick, extract minim DNA efficiently.
Through different condition combination, serial simultaneous test and the test of a large amount of on-the-spot case sample, we find: 1. protease digestion, 70-100 temperature are bathed, PEG-ethanol magnetic bead can high efficiency extraction minim DNA in conjunction with the combination of liquid 3; 2. protease digestion, temperature bath process can efficiently discharge cast-off cells DNA, structural integrity, are easily combined with magnetic bead in PEG-ethanol system; 3. the use of PEG-ethanol articulated system not only promotes that magnetic bead is to the efficient adsorption of DNA, and ensure that DNA structure is relatively complete, obtains the needs that the DNA fragmentation that is of convenient length can meet 16 STR site composite amplifications in case detection.Reagent extracts case trace sample DNA can either reduce cost, easy operation, without the need to the cross-reference of plurality of reagents box and multiple method, and also highly sensitive, method is simple, decreases the dependence to technician's experience, achieves the lifting that case is detected as power.
The invention provides a kind of reagent extracting case trace sample DNA, it consists of suspension containing magnetic beads; Lysate, it consists of the Tris-HCl damping fluid of pH=8.0; In conjunction with liquid I, it consists of the PEG of 20-50%w/v, the MW=8000 of PEG; In conjunction with liquid II, it consists of ethanol; Scavenging solution, it consists of the ethanolic soln of 50-80%v/v.
Of the present inventionly can replace ethanol by the one in methyl alcohol and propyl alcohol, Virahol in conjunction with liquid II.
The inventive method can extract DNA from micro-case sample simple and effective.
Micro-case sample of the present invention is the biological material containing micro-case trace sample that criminal-scene is left over.
The filter membrane that the carrier having shifted case trace sample of the present invention obtains for the clothing, rope, electric wire, waddy etc. left over biomass cells extraction apparatus absorption criminal-scene.
The carrier having shifted case trace sample of the present invention is the glued membrane that glues clothing, rope, electric wire, waddy, bottleneck, the fingerprint etc. on various handle or handle, glass or desktop etc. left over enchashment field with the micro-sample Adhesive-fetch device of case, scotch tape and obtain or scotch tape.
Filter paper, cotton swab, yarn etc. that fingerprint etc. on the clothing that the carrier having shifted case trace sample of the present invention is filter paper, the sassafras scene of wiping such as cotton swab, yarn is left over, rope, electric wire, waddy, bottleneck, various handle or handle, glass or desktop etc. obtains.
For lysate of the present invention, in conjunction with liquid I, be not particularly limited in conjunction with liquid II, scavenging solution, the lysate that can adopt for this area, in conjunction with liquid I, in conjunction with liquid II, scavenging solution.
Major advantage of the present invention is:
1. the inventive method associated proteins enzymic digestion, the bath of 70-100 degree temperature can fully cracking case trace sample cast-off cells, and the DNA structure of release is relatively complete, in PEG-ethanol system easy be combined with magnetic bead and;
2. in reagent composition, PEG-ethanol not only can remove protein better in conjunction with the existence of liquid, and magnetic bead can be well-dispersed in conjunction with in liquid when adsorption of DNA, achieves magnetic bead to the efficient adsorption in conjunction with DNA in liquid;
3.PEG-ethanol makes the DNA length extracted be applicable in conjunction with the use of liquid, can meet the needs of 16 STR site composite amplifications in case detection, greatly reduces and loses peak, amplification inequality phenomenon.
4. this extracting method decreases the dependency to operator's experience, easy and simple to handle, good stability;
5., without the need to using the organic solvent that the toxicity such as phenol, chloroform is large, security is good;
6. highly sensitive, extract fast, general operation can complete at 1 ~ 1.5 hour, and can be used for the DNA extraction of Automation workstation.
Accompanying drawing explanation
The Multiplex STR amplification collection of illustrative plates of the minim DNA that the belt that Fig. 1 criminal-scene is left over extracts.
The DNA Multiplex STR amplification collection of illustrative plates that Figure 21 μ L extracts through 60 times of dilution fresh bloods.
Figure 31 μ L is through 60 times of dilution fresh bloods, and DNA Multiplex STR amplification collection of illustrative plates is extracted in control experiment 2.
Figure 41 μ L is through 60 times of dilution fresh bloods, and DNA Multiplex STR amplification collection of illustrative plates is extracted in control experiment 3.
Figure 51 μ L is through 60 times of dilution fresh bloods, and DNA Multiplex STR amplification collection of illustrative plates is extracted in control experiment 4.
Figure 61 μ L is through 60 times of dilution fresh bloods, and DNA Multiplex STR amplification collection of illustrative plates is extracted in control experiment 5.
Figure 71 μ L is through 60 times of dilution fresh bloods, and DNA Multiplex STR amplification collection of illustrative plates is extracted in control experiment 6.
Figure 81 μ L is through 60 times of dilution fresh bloods, and DNA Multiplex STR amplification collection of illustrative plates is extracted in control experiment 7.
Figure 91 μ L is through 60 times of dilution fresh bloods, and DNA Multiplex STR amplification collection of illustrative plates is extracted in control experiment 8.
Figure 10 is the slippers that criminal-scene is left over, and replaces the Multiplex STR amplification collection of illustrative plates in conjunction with liquid II with methyl alcohol.
Figure 11 is the toothbrush that criminal-scene is left over, and replaces the Multiplex STR amplification collection of illustrative plates in conjunction with liquid II with propyl alcohol.
Figure 12 is the porcelain cup drunk water that criminal-scene is left over, and replaces the Multiplex STR amplification collection of illustrative plates in conjunction with liquid II with Virahol.
Embodiment
Embodiment one extracts the minim DNA on belt hook that criminal-scene leaves over.
(1) test kit prepares
Preparation utilizes magnetic bead to extract the test kit of case trace sample DNA, comprising:
1mL silanization magnetic bead solution (0.1mg/ μ L), Wuhan Wawasye Technology Development Co., Ltd.'s commercialized product or Qiagen company buy;
100mL lysate, its component is the Tris-HCl damping fluid of pH=8.0;
100mL is in conjunction with liquid I, and its volume components is than the PEG (molecular weight 8000) for 20-50%;
20mL is in conjunction with liquid II, and its component is ethanol;
100mL scavenging solution, its component to be volume ratio be 80% ethanolic soln.
(2) utilize the belt hook cast-off cells DNA that test kit extraction criminal-scene is left over, extraction step is as follows:
1) utilize case trace sample Adhesive-fetch device repeatedly ixoderm bracelet repeatedly, glued membrane is put into 1.5mL centrifuge tube;
2) add 6 μ L Proteinase Ks (10mg/mL), 350 μ L lysates, whirlpool shook for 2 seconds, placed couveuse 56 degree and hatched 40 minutes;
3) taken off from couveuse by centrifuge tube, whirlpool shook for 2 seconds, and after placement couveuse 70-100 degree hatches 8 minutes, 13000 leave the heart 3 minutes, and supernatant is transferred to another 1.5mL centrifuge tube;
4) add 400 μ L in conjunction with liquid I, 80 μ L in conjunction with liquid II and 20 μ L suspension containing magnetic beads, whirlpool shook for 2 seconds, placed room temperature 10 minutes;
5) by centrifuge tube as magnetic frame upper 2 minute, inhale and abandon supernatant and stay magnetic bead, add 300 μ L scavenging solutions, whirlpool shook for 2 seconds, placed room temperature 2 minutes;
6) repeating step 4 twice;
7) by centrifuge tube as magnetic frame upper 2 minute, inhale and abandon supernatant and stay magnetic bead, centrifuge tube is opened and is placed room temperature 5 minutes;
8) add 30 μ L ultrapure waters, whirlpool shook for 2 seconds, placed couveuse 80 degree and hatched 10 minutes, and middle mixing magnetic bead 2 times, centrifuge tube is placed in magnetic frame, and gained supernatant is DNA solution.
(3) pcr amplification and electrophoresis
Amplification kit is the sinofiler test kit of ABI company
Amplification program is as follows:
Electrophoresis equipment is 3100 type sequenators of ABI company
Loading system: 2 μ L amplified productions+20 μ L methane amide (adding Liz)
Other steps: operate in strict accordance with 3100 type sequenator specification sheetss.
Fig. 1 extracts the detected result that this criminal-scene leaves over belt DNA, 16 STR sites are complete, peak height, all more than 200, meets the requirement of case detection to 16 STR Locus Analysis in Shoots, and the tool when extracting micro-case detection DNA further illustrating this reagent and present method has great advantage.
Embodiment two control experiment
With 1 μ L through the blood of 60 times of normal saline dilutions for analog sample, replace embodiment one cast-off cells glued membrane, amplification and electrophoresis are with embodiment one, and it is as follows that design trace sample extracts control series test:
Controlled trial 1 extraction step 1) in glued membrane replaced by 1 μ L analog sample, other steps are constant;
Controlled trial 2 step 2) in do not add proteolytic enzyme, other are with controlled trial 1;
Controlled trial 3 step 3) middle incubation temperature is less than 70 degree, and other are with test 1;
Controlled trial 4 step 4) in remove ethanol in conjunction with in liquid system, other with test 1;
Controlled trial 5 step 4) in remove PEG in conjunction with in liquid system, other with test 1;
Controlled trial 6 is through step 1), 2), 3), supernatant with QiagenM48 magnetic bead kit extract DNA;
Controlled trial 7 is without step 1), 2), 3), with M48 magnetic bead reagent extract DNA;
Controlled trial 8 is through step 1), 2), 3, supernatant uses Microcon-100 purification column purified concentration DNA instead.
All carry out multiplex PCR composite amplification with the ID test kit of ABI company with the system of 4 μ L template+6 μ LMix through testing the DNA solution extracted above, and carry out electrophoresis detection with identical system and condition, acquired results is as shown in Fig. 2-Fig. 9.Wherein, Fig. 2 is the result of control series test 1: 16 STR sites are complete, peak height is all more than 800, meet the requirement of case detection to 16 STR Locus Analysis in Shoots, illustrate that cast-off cells DNA can efficiently discharge after protease digestion, temperature bath process, in PEG-ethanol articulated system, efficient adsorption is in magnetic bead surfaces.Fig. 3 is the result of control series test 2: the peak in CSF1PO site is lost, the peak in D12S391 and D3S1350 site has height to have the end, amplification inequality phenomenon is obvious, D7S820 and D18S51 site is a many peak, namely have assorted peak, these all have a strong impact on the somatotype to detected result, do not meet the requirement of case detection to 16 STR Locus Analysis in Shoots, test illustrates that cast-off cells are without protease digestion, and DNA release is incomplete or rupture.Fig. 4 is the result of control series test 3: small segment effect is better as peak, D8S1179 and D19S433 site all goes out, the peak of sheet degree is too low, as D7S820 below 50, and there is assorted peak phenomenon as D13S317 site, illustrate that DNA is poor in conjunction with magnetic bead efficiency in PEG-ethanol system, controlled trial 2 and 3 illustrates protease digestion, 70-100 degree temperature bath cell high-efficient released dna, and DNA structure is stablized, and is beneficial to and is combined with magnetic bead in PEG-ethanol system.Fig. 5 is the result of control series test 4: 16 sites all do not go out, Fig. 6 is the result of control series test 5, site D8S1179, D5S818, vWA and D8S1043 are only gone out, but peak is below 100, D8S1043 has assorted peak phenomenon, do not meet the requirement of case detection to 16 STR Locus Analysis in Shoots, ethanol and PEG must be there is in controlled trial 4 and 5 description taken in conjunction liquid system simultaneously, ensure DNA and magnetic bead joint efficiency high, DNA structure is relatively complete, obtains the needs that the DNA fragmentation that is of convenient length can meet 16 STR site composite amplifications in case detection.Fig. 7 is the result of control series test 6, Fig. 8 is the result of control series test 7, and the two result is more or less the same, and 16 sites all do not go out, peak height is below 200, and assorted peak is also many, affects the interpretation of result, do not meet the requirement of case detection to 16 STR Locus Analysis in Shoots, Fig. 9 is the result of control series test 8, similar with Fig. 6, only gone out the peak in 6 sites, ratio of peak is lower.Lose peak and show DNA structure destroyed or DNA extraction amount is low, enough, to be of convenient length the amplification of satisfied 16 STR sites DNA can not be obtained.Test 7 illustrate the DNA structure that guanidinesalt magnetic bead (M48 reagent) articulated systems may destroy or joint efficiency low.Test 6,8 interpret sample are after protease digestion, 70-100 degree temperature bath DNA efficiently discharge, PEG-ethanol magnetic bead articulated system not only efficient adsorption DNA and also ensure that DNA structure stablize, be better than guanidinesalt magnetic bead (M48 reagent) articulated system and guanidinesalt purification column articulated system (Microcon-100 reagent)---these two kinds of systems are micro-sample DNA extraction common agents.To sum up the Comparative result of 8 control series tests is known, the DNA solution extracted with reagent of the present invention and method can obtain complete STR somatotype, and STR peak height is all more than 200, and when cracking condition and articulated system change, all do not obtain complete STR somatotype, the peak-to-peak value gone out is substantially below 200.
Embodiment three changes methyl alcohol and propyl alcohol, Virahol in conjunction with the ethanol of liquid II
Change methyl alcohol and propyl alcohol, Virahol by embodiment one respectively in conjunction with the ethanol of liquid II, extract the DNA on the slippers in on-the-spot case, toothbrush, the porcelain cup that drinks water with paramagnetic particle method respectively;
Except 80 μ L in extraction step 3 change into except 80 μ L methyl alcohol and propyl alcohol, Virahol in conjunction with liquid II respectively, other operations are with embodiment one.
Figure 10 is the slippers that criminal-scene is left over, and replaces the Multiplex STR amplification collection of illustrative plates in conjunction with liquid II with methyl alcohol; Figure 11 is the toothbrush that criminal-scene is left over, and replaces the Multiplex STR amplification collection of illustrative plates in conjunction with liquid II with propyl alcohol; Figure 12 is the porcelain cup drunk water that criminal-scene is left over, and replaces the composite S TR in conjunction with liquid II with Virahol.After changing methyl alcohol, propyl alcohol and Virahol into respectively by the known ethanol in conjunction with liquid II of Figure 10, Figure 11, Figure 12, it is complete that detected result through same procedure extraction criminal-scene biological material is 16 STR sites, peak height is all more than 200, meet case and detect requirement to 16 STR Locus Analysis in Shoots, illustrate and methyl alcohol, propyl alcohol and Virahol can be used to replace in conjunction with II in this extracting method and this test kit.
Reference:
1. pungent army equality, the process of the difficult sample product of medical science and detection method research, criminal technique, 2001,6.
2. Wang Qin etc., DNA-STR somatotype inspection human body case trace sample 2 example, criminal technique, 2004,5.
3. Wang Qin etc., the fluorescent mark STR somatotype of human body case trace sample, law and medical journal, 2004,11 (1).
4. a celebrating rosy clouds etc., extract trace sample DNA and crack murder case 1 example, Journal of Forensic Sciences, 2007,2,1 (23).
5. Wang Yu is good for, case trace sample minim DNA detection scheme pre-test on clothing, criminal technique, 2007,2.
6. Zhang Zhi is superfine, the finishing of magnetic silica microballoon and the application in Plant Genome nucleic acid purification thereof, analytical chemistry research report, 2007,1 (35).
7. the Cong Xian tinkling of pieces of jade etc., adopt silicon dioxide microsphere method to reclaim the experimental study of DNA, China Immunology Journal, 2005,5.
Claims (4)
1. one kind is improved the method for cast-off cells DNA extraction efficiency, it is characterized in that being made up of following steps: with the sample of Proteinase K pack processing containing cast-off cells, then treated sample is hatched 3-8 minute in 70-100 degree Celsius, centrifuging and taking Sample supernatants, in PEG-ethanol binding buffer system, allow the DNA in supernatant be combined with magnetic bead, be separated and obtain DNA, wherein in above-mentioned steps, not using guanidine salt reagent.
2. method according to claim 1, wherein ethanol methyl alcohol, propyl alcohol or Virahol substitute.
3. method according to claim 1, is characterized in that, described magnetic bead is through silanization treatment.
4. extract a method of case trace sample DNA, it is characterized in that carrying out according to the following steps:
1) may be placed in centrifuge tube containing the carrier of cast-off cells by what shifted, and add lysate and Proteinase K, whirlpool concussion mixing, centrifuge tube to be placed on couveuse 56 degrees Celsius and to hatch 40-60 minute;
2) centrifuge tube whirlpool concussion mixing, to be placed on couveuse 70-100 degree Celsius hatch 3-8 minute after, centrifugal 3 minutes of 13000rpm;
3) shift supernatant, add suspension containing magnetic beads, in conjunction with liquid I with in conjunction with liquid II, piping and druming mixing, forms the suspension liquid of solid-liquid homodisperse;
4) magnetic bead adsorbing DNA is isolated by magnetic frame from the suspension liquid of solid-liquid homodisperse;
5) scavenging solution washing is adsorbed with magnetic bead 2-3 time of DNA, is washed away by the impurity on magnetic bead;
6) DNA on ultrapure water desorption magnetic bead, obtains pure concentrated DNA solution;
The Tris-HCl consisting of pH=8.0 of described lysate, the EDTA of pH=8.0,50-80mmol/L;
Described is the MW=8000 of the PEG of 20-50%w/v, PEG in conjunction with liquid I;
Described is ethanol, methyl alcohol, propyl alcohol or Virahol in conjunction with liquid II;
Described scavenging solution is the ethanolic soln of 50-80%v/v.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110148869.7A CN102229927B (en) | 2011-06-03 | 2011-06-03 | Improve method and the reagent of rate of extraction of DNAs of castoff cells in case trace sample |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110148869.7A CN102229927B (en) | 2011-06-03 | 2011-06-03 | Improve method and the reagent of rate of extraction of DNAs of castoff cells in case trace sample |
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| Publication Number | Publication Date |
|---|---|
| CN102229927A CN102229927A (en) | 2011-11-02 |
| CN102229927B true CN102229927B (en) | 2016-03-23 |
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| CN103622701B (en) * | 2012-08-23 | 2018-08-10 | 聂棱 | Silanization nano-magnetic powder shows fingerprint method |
| CN104830833B (en) * | 2015-03-23 | 2018-08-24 | 上海惠文生物技术有限公司 | A kind of method of paramagnetic particle method combination centrifuge shield extraction DNA |
| CN106701737B (en) * | 2015-11-17 | 2020-07-31 | 安诺优达基因科技(北京)有限公司 | High-efficiency DNA purification magnetic bead reagent with fragment selective purification capability |
| CN108893465A (en) * | 2018-07-25 | 2018-11-27 | 古蔺县公安局 | Magnetic bead is used to show the purposes and impression of the hand trace DNA extraction method of impression of the hand trace |
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| WO1995013368A1 (en) * | 1993-11-11 | 1995-05-18 | Medinnova Sf | Isolation of nucleic acid |
| CN101397588A (en) * | 2008-11-06 | 2009-04-01 | 昆明理工大学 | Reagent and kit for acquisition and recovery trace amount DNA and use method |
| CN101613697A (en) * | 2009-08-05 | 2009-12-30 | 公安部物证鉴定中心 | A kind of method of extracting purify DNA |
| CN102220310A (en) * | 2009-08-05 | 2011-10-19 | 公安部物证鉴定中心 | Method for extracting and purifying spittle DNA |
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| AU2003217552A1 (en) * | 2002-02-19 | 2003-09-09 | Choicepoint Asset Company | Selective extraction of dna from groups of cells |
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|---|---|---|---|---|
| WO1995013368A1 (en) * | 1993-11-11 | 1995-05-18 | Medinnova Sf | Isolation of nucleic acid |
| CN101397588A (en) * | 2008-11-06 | 2009-04-01 | 昆明理工大学 | Reagent and kit for acquisition and recovery trace amount DNA and use method |
| CN101613697A (en) * | 2009-08-05 | 2009-12-30 | 公安部物证鉴定中心 | A kind of method of extracting purify DNA |
| CN102220310A (en) * | 2009-08-05 | 2011-10-19 | 公安部物证鉴定中心 | Method for extracting and purifying spittle DNA |
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