CN105624149A - Kit and method for extracting and purifying DNA (deoxyribonucleic acid) in untouchable specimen - Google Patents
Kit and method for extracting and purifying DNA (deoxyribonucleic acid) in untouchable specimen Download PDFInfo
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Abstract
The invention provides a kit and method for effectively extracting and purifying DNA (deoxyribonucleic acid) in an untouchable specimen. The kit is composed of a digestive solution, an adsorptive solution, a washing solution 1, a washing solution 2, a washing solution 3, a magnetic bead suspension and an eluting solution. The method for extracting DNA by using the kit comprises the following steps: exfoliated cell digestion, DNA adsorption, magnetic separation, gradient washing and eluting. The kit has high extraction sensitivity, and can effectively complete the extraction of DNA in an untouchable specimen. The kit has the advantages of high impurity interference resistance and high purity, and has favorable effects on extracting DNA in the specimen containing dust, clay and other impurities. The kit is simple to operate, and can be matched with an automatic extraction apparatus.
Description
Technical field
The present invention relates to a kind of test kit and method that can effectively carry out DNA extraction in high-leveled and difficult contact sample, belong to biological and technical field of medical examination.
Background technology
DNA inspection technology is the important composition of current forensic medical examination technology, by the DNA sample of criminal-scene collection is carried out sequencing analysis, utilizing STR(STR) typing method can carry out individual identification accurately, thus providing strong evidence support for cracking of cases and court judgment. Under normal circumstances, forensic dna inspection comprises several steps such as the collection of DNA sample, DNA extraction and purification, amplification and order-checking, and can wherein the extraction of DNA and purification be the premises of subsequent survey, play vital effect to obtain effective STR typing. For the case sample that DNA content is higher, for instance ecchymosis, tissue, seminal stain, saliva etc., conventional method (such as Chelex100 method etc.) is adopted can effectively to realize extraction and the purification of DNA. But along with the development of forensic dna technology, the demand of DNA extraction purification in contact sample in case is got more and more. So-called contact sample refers to the sample that the exfoliative cyte by being retained on object after contact skin object are constituted, for instance fingerprint, palmmprint, glove, contact medicated clothing etc. Contact sample changes various in criminal-scene, and its DNA content also differs widely. Such as the single piece of fingerprint that scene is left over, DNA content will far fewer than the vestige repeatedly touched. As suspect is with glove to commit a crime, the exfoliative cyte left over through glove touching object secondary transferring are then more rare. It addition, inevitably adulterate various impurity (such as dust, earth, rust etc.) from collection in worksite contact sample. At present, single piece of fingerprint, secondary transferring sample and high impurity sample are all referred to as high-leveled and difficult contact sample, adopt conventional method, even adopt some traditional extraction test kits all cannot effectively carry out DNA extraction and purification. And these high-leveled and difficult contact samples often vital source of evidence in cracking of cases, it is impossible to effectively it being carried out DNA extraction, to directly result in high-leveled and difficult contact sample DNA recall rate very low, it is impossible to meets growing case inspection demand.
For case above, it is necessary to develop a kind of high DNA extraction purification kit extracting sensitivity and strong purification capacity that has concurrently, and the effective ways of DNA extraction purification in high-leveled and difficult contact sample can be completed according to the foundation of this test kit.
Summary of the invention
It is an object of the invention to provide a kind of test kit and method that can effectively carry out DNA extraction in high-leveled and difficult contact sample, it is highly sensitive that this test kit has extraction, and anti-impurity effect ability is strong, it is easy to the advantages such as operation.
The test kit of the present invention and extracting method adopt nanometer magnetic bead as the medium extracting DNA, low based on DNA content in high-leveled and difficult contact sample and that impurity content is high two difficult points, devise extraction reagent system and correlation method, thus realizing effectively obtaining DNA sample from high-leveled and difficult contact sample. First technological innovation of the present invention is to optimize DNA absorption system to improve the magnetic bead absorbability to DNA, thus improving extraction sensitivity. Another important technology innovation of the present invention is to devise brand-new washing system and washing methods for the magnetic bead after adsorption of DNA. This washing methods impurity (such as albumen, metal ion, solid particle etc.) except effectively can removing DNA, more importantly the cleaning mixture by adopting polarity and ionic strength to successively decrease, by the mode of sequential purge, the short segment invalidity DNA eluting on magnetic bead will be adsorbed on, retain the DNA long fragment comprising STR information. Advantage of this is that, in subsequent PCR amplification reacts, owing to eliminating short segment invalidity DNA to the PCR Competition reacted, STR effective dna fragment can be made fully to expand, the amplification quantity improving effective fragment can be greatly enhanced the useful signal of final capillary electrophoresis, so that high-leveled and difficult contact sample DNA recall rate is effectively promoted.
A kind of test kit for high-leveled and difficult contact sample DNA extraction purification described in the present invention, it is characterised in that: it is made up of Digestive system, adsorption liquid, cleaning mixture 1, cleaning mixture 2, cleaning mixture 3, magnetic bead suspension and eluent seven part;
Wherein, described Digestive system consist of guanidinium isothiocyanate (2.0 ~ 5.0M), guanidine hydrochloride (0.1 ~ 0.5M), TrisHCl(0.02 ~ 0.04M), Tween80(7.0 �� 10-3~1.4��10-2M), EDTA(2.0 �� 10-3~5.0��10-3M), SDS(3.0 �� 10-3~1.5��10-2M);
Described adsorption liquid consists of guanidinium isothiocyanate (2.0 ~ 5.0M), TrisHCl(0.02 ~ 0.04M), ethylene glycol (1.5 ~ 2.5M);
Described cleaning mixture 1 consist of guanidinium isothiocyanate (2.0 ~ 5.0M), TrisHCl(0.02 ~ 0.04M), ethylene glycol (3.0 ~ 4.0M);
Described cleaning mixture 2 consist of dehydrated alcohol: isopropanol: water=7:1:2(volume ratio), add NaCl be 2.0M to concentration;
Described cleaning mixture 3 consist of dehydrated alcohol: isopropanol: water=6:2:2(volume ratio), add NaCl be 0.5M to concentration;
Described magnetic bead suspension is the super-paramagnetism nano magnetic bead of size 100 ~ 2000nm, is dispersed in high purity water, and concentration is 9%(mass percent);
Described eluent is high purity water.
Described adsorption liquid its act as the absorption combining environmental that DNA and magnetic bead are provided, the adsorption liquid system of the present invention is characterized by adding ethylene glycol, the polarity of Optimum Regulation adsorption liquid system, strengthens the adsorption between magnetic bead and DNA.
Described cleaning mixture its act as by washing operation, remove the impurity except DNA of magnetic bead absorption. The cleaning mixture of the present invention is characterised by, the cleaning mixture system of polarity and ionic strength graded by optimizing design, realizing on the basis that impurity fully washs, furthermore achieved that the selective elution to short segment invalidity DNA, thus improving the extraction quality of effective dna. Cleaning mixture system is divided into the different composition of cleaning mixture 1, cleaning mixture 2, cleaning mixture 3 three.
Described magnetic bead suspension is super-paramagnetism nano magnetic bead, and size, between 100 ~ 2000nm, is dispersed in high purity water, and concentration is 9%(mass percent).
The preparation method of a kind of test kit for high-leveled and difficult contact sample DNA extraction purification described in the present invention, comprises the following steps:
Weighing the reagent that preparation Digestive system, adsorption liquid, cleaning mixture 1, cleaning mixture 2, cleaning mixture 3, magnetic bead suspension and eluent relate in proportion respectively, compound method routinely is mix homogeneously respectively.
The using method of the test kit for high-leveled and difficult contact sample DNA extraction purification of the present invention, comprises the following steps:
1) the contact sample taking suitable size is put in centrifuge tube, adds 100 ~ 1000 �� l Digestive systems, adds the E.C. 3.4.21.64 aqueous solution of 10mg/ml according to the ratio of volume ratio 10:1 ~ 20:1; Overturn after mixing, be placed in 56 DEG C of temperature baths 0.5 hour ~ 6 hours;
2) after temperature bath, centrifugal 12000rpm, 3 minutes; Take out supernatant and be placed in new centrifuge tube, in supernatant, add the adsorption liquid of 1 ~ 10 times of supernatant volume, add 5 ~ 30 �� l magnetic bead suspensions; On convertible blending instrument, room temperature mixes absorption 10 ~ 20 minutes continuously;
3) centrifuge tube is placed on magnetic frame and carries out Magneto separate, inhale liquid in pipe abandon, retain magnetic bead;
4) in centrifuge tube, add 150 ~ 400 �� l cleaning mixture 1, cover centrifuge tube lid, put back to magnet stand immediately after vortex vibration mixing, inhale and abandon liquid;
5) in centrifuge tube, add 150 ~ 500 �� l cleaning mixture 2, cover centrifuge tube lid, put back to magnet stand immediately after vortex vibration mixing, inhale and abandon liquid;
6) in centrifuge tube, add 150 ~ 500 �� l cleaning mixture 3, cover centrifuge tube lid, put back to magnet stand immediately after vortex vibration mixing, inhale and abandon liquid, drying at room temperature of uncapping 5 ~ 15 minutes;
7) add 10 ~ 30 �� l eluents, vortex vibration mixing after be put into immediately on magnet stand, treat that magnetic bead is adsorbed onto on centrifuge tube tube wall completely, sucking-off solution be put in another centrifuge tube preserves stand-by.
The positive effect of the present invention is in that: DNA extraction is highly sensitive, it is possible to the effective extraction purification completing DNA from only high-leveled and difficult contact sample containing pettiness amount DNA. Anti-impurity interference performance is strong, the sample containing the impurity such as dust, earth is extracted DNA effective, and purity is high. Easy and simple to handle, it is possible to extract instrument with automatization and coordinate, improve work efficiency.
Accompanying drawing explanation
Fig. 1. the STR spectrogram of the DNA sample extracted in embodiment 1;
Fig. 2. the STR spectrogram of the DNA sample extracted in embodiment 2.
Detailed description of the invention
By following example, the present invention is described further, it should be appreciated that following example are exemplary, rather than restrictive, according to the technical scheme of foregoing invention and practical situation, concrete implementation can be determined.
Embodiment 1
The present invention can effectively carry out the test kit of DNA extraction purification in high-leveled and difficult contact sample, forms including Digestive system, adsorption liquid, cleaning mixture 1, cleaning mixture 2, cleaning mixture 3, magnetic bead suspension and eluent seven part;
Described Digestive system consist of guanidinium isothiocyanate (2.0M), guanidine hydrochloride (0.1M), TrisHCl(0.02M), Tween80(7.0 �� 10-3M), EDTA(2.0 �� 10-3M), SDS(3.0 �� 10-3M);
Described adsorption liquid consists of guanidinium isothiocyanate (2.0M), TrisHCl(0.02M), ethylene glycol (1.5M);
Described cleaning mixture 1 consist of guanidinium isothiocyanate (2.0M), TrisHCl(0.02M), ethylene glycol (3.0M);
Consisting of of described cleaning mixture 2, configures by volume, dehydrated alcohol: isopropanol: water=7:1:2; Adding NaCl is 2.0M to concentration;
Consisting of of described cleaning mixture 3, configures by volume, dehydrated alcohol: isopropanol: water=6:2:2; Adding NaCl is 0.5M to concentration;
Described magnetic bead suspension is super-paramagnetism nano magnetic bead, and size, between 100 ~ 2000nm, is dispersed in high purity water, and concentration is 9%(mass percent);
Described eluent is high purity water;
Weighing the reagent that preparation Digestive system, adsorption liquid, cleaning mixture 1, cleaning mixture 2, cleaning mixture 3, magnetic bead suspension and eluent relate in proportion respectively, compound method routinely is mix homogeneously respectively.
Embodiment 2
A kind of described in the present invention can effectively carry out the test kit of DNA extraction purification in high-leveled and difficult contact sample, forms including Digestive system, adsorption liquid, cleaning mixture 1, cleaning mixture 2, cleaning mixture 3, magnetic bead suspension and eluent seven part:
Described Digestive system consist of guanidinium isothiocyanate (5.0M), guanidine hydrochloride (0.5M), TrisHCl(0.04M), Tween80(1.4 �� 10-2M), EDTA(5.0 �� 10-3M), SDS(1.5 �� 10-2M);
Described adsorption liquid consists of guanidinium isothiocyanate (5.0M), TrisHCl(0.04M), ethylene glycol (2.5M);
Described cleaning mixture 1 consist of guanidinium isothiocyanate (5.0M), TrisHCl(0.04M), ethylene glycol (4.0M);
Consisting of of described cleaning mixture 2, configures by volume, dehydrated alcohol: isopropanol: water=7:1:2; Adding NaCl is 2.0M to concentration;
Consisting of of described cleaning mixture 3, configures by volume, dehydrated alcohol: isopropanol: water=6:2:2; Adding NaCl is 0.5M to concentration;
Described magnetic bead suspension is super-paramagnetism nano magnetic bead, and size, between 100 ~ 2000nm, is dispersed in high purity water, and concentration is 9%(mass percent);
Described eluent is high purity water;
Weighing the reagent that preparation Digestive system, adsorption liquid, cleaning mixture 1, cleaning mixture 2, cleaning mixture 3, magnetic bead suspension and eluent relate in proportion respectively, compound method routinely is mix homogeneously respectively.
Embodiment 3
Can effectively carry out the test kit of DNA extraction purification in high-leveled and difficult contact sample described in the present invention, form including Digestive system, adsorption liquid, cleaning mixture 1, cleaning mixture 2, cleaning mixture 3, magnetic bead suspension and eluent seven part;
Described Digestive system consist of guanidinium isothiocyanate (3.0M), guanidine hydrochloride (0.3M), TrisHCl(0.03M), Tween80(1.0 �� 10-2M), EDTA(3.5 �� 10-3M), SDS(9 �� 10-3M);
Described adsorption liquid consists of guanidinium isothiocyanate (3.0M), TrisHCl(0.03M), ethylene glycol (2.0M);
Described cleaning mixture 1 consist of guanidinium isothiocyanate (3.5M), TrisHCl(0.03M), ethylene glycol (3.5M);
Consisting of of described cleaning mixture 2, configures by volume, dehydrated alcohol: isopropanol: water=7:1:2; Adding NaCl is 2.0M to concentration;
Consisting of of described cleaning mixture 3, configures by volume, dehydrated alcohol: isopropanol: water=6:2:2; Adding NaCl is 0.5M to concentration;
Described magnetic bead suspension is super-paramagnetism nano magnetic bead, and size, between 100 ~ 2000nm, is dispersed in high purity water, and concentration is 9%(mass percent);
Described eluent is high purity water;
Weighing the reagent that preparation Digestive system, adsorption liquid, cleaning mixture 1, cleaning mixture 2, cleaning mixture 3, magnetic bead suspension and eluent relate in proportion respectively, compound method routinely is mix homogeneously respectively.
Experimental example 1
Use the DNA extraction kit according to embodiment 1 configuration, DNA in the cotton swab after wiping fingerprint carried out extraction purification, comprises the following steps:
1, the cotton swab head Cotton Gossypii after wiping fingerprint is peeled off, be placed in 1.5ml centrifuge tube, add Digestive system 120ul, add the Proteinase K Solution of 6ul, 10mg/ul. Overturn after mixing, be placed in 56 DEG C of temperature baths 1 hour.
2, after temperature bath, centrifugal 12000rpm, 3 minutes. Take out supernatant and be placed in new centrifuge tube, in supernatant, add the adsorption liquid of 3 times of supernatant volumes, add 10ul magnetic bead suspension. On convertible blending instrument, room temperature mixes absorption 15 minutes continuously.
3, centrifuge tube is placed on magnetic frame and carries out Magneto separate, inhale liquid in pipe abandon, retain magnetic bead.
4, in centrifuge tube, add 150ul cleaning mixture 1, cover centrifuge tube lid, put back to magnet stand immediately after vortex vibration mixing, inhale and abandon liquid.
5, in centrifuge tube, add 150ul cleaning mixture 2, cover centrifuge tube lid, put back to magnet stand immediately after vortex vibration mixing, inhale and abandon liquid.
6, in centrifuge tube, add 150ul cleaning mixture 3, cover centrifuge tube lid, put back to magnet stand immediately after vortex vibration mixing, inhale and abandon liquid, uncap dry 5 minutes.
7, add 10ul eluent, vortex vibration mixing after be put into immediately on magnet stand, treat that magnetic bead is adsorbed onto on centrifuge tube tube wall completely, sucking-off solution be put in another centrifuge tube preserves stand-by.
8, take the DNA solution after purification and carry out pcr amplification and electrophoresis, it is thus achieved that STR typing spectrogram as shown in Figure 1.
Experimental example 2
Use the DNA extraction kit according to embodiment 2 configuration, DNA in the diaphragm after the viscous print of exfoliative cyte Adhesive-fetch device carried out extraction purification, comprises the following steps:
1, exfoliative cyte Adhesive-fetch device surface membrane tweezers are torn, be placed in 1.5ml centrifuge tube, add Digestive system 240ul, add the Proteinase K Solution of 20ul, 10mg/ul. Overturn after mixing, be placed in 56 DEG C of temperature baths 0.5 hour.
2, after temperature bath, centrifugal 12000rpm, 3 minutes. Take out supernatant and be placed in new centrifuge tube, in supernatant, add the adsorption liquid of 5 times of supernatant volumes, add 15ul magnetic bead suspension. On convertible blending instrument, room temperature mixes absorption 20 minutes continuously.
3, centrifuge tube is placed on magnetic frame and carries out Magneto separate, inhale liquid in pipe abandon, retain magnetic bead;
4, in centrifuge tube, add 200ul cleaning mixture 1, cover centrifuge tube lid, put back to magnet stand immediately after vortex vibration mixing, inhale and abandon liquid;
5, in centrifuge tube, add 200ul cleaning mixture 2, cover centrifuge tube lid, put back to magnet stand immediately after vortex vibration mixing, inhale and abandon liquid;
6, in centrifuge tube, add 200ul cleaning mixture 3, cover centrifuge tube lid, put back to magnet stand immediately after vortex vibration mixing, inhale and abandon liquid, uncap dry 8 minutes;
7, add 20ul eluent, vortex vibration mixing after be put into immediately on magnet stand, treat that magnetic bead is adsorbed onto on centrifuge tube tube wall completely, sucking-off solution be put in another centrifuge tube preserves stand-by;
8, take the DNA solution after purification and carry out pcr amplification and electrophoresis, it is thus achieved that STR typing spectrogram as shown in Figure 2.
For test kit and the method prominent effect in high-leveled and difficult contact sample DNA extraction of the present invention are described, this experimental example selects the traditional method chelex-100 method method as a comparison of current DNA extraction, carries out the experiment of high-leveled and difficult contact sample DNA extraction with the test kit of the present invention simultaneously.
Chelex-100 method extracts DNA experimental procedure:
(1) sample is through digestion, and released dna is in solution.
(2) adding 5%chelex-100 solution, repeatedly shake, 56 DEG C are incubated 30 minutes
(3) concussion solution mixing, centrifugal 3 minutes of 13000rpm, take supernatant and check order for pcr amplification and STR.
In experiment, test kit configures according to formula in embodiment 1,2,3, and DNA sample chooses same sample DNA solution after digestion process. Pcr amplification is all identical with STR deposition condition. Experimental result is table 1 such as. Be can be seen that high-leveled and difficult contact sample DNA extraction is had significant advantage by the test kit of the present invention and extracting method by experimental result.
Table 1. Different Extraction Method Contrast on effect to high-leveled and difficult contact sample DNA extraction
| Chelex-100 | Embodiment 1 test kit | Embodiment 2 test kit | Embodiment 3 test kit | |
| Cotton swab fingerprint | STR does not detect | STR bit point is complete, peak averaging 1000 | STR bit point is complete, peak averaging 900 | STR bit point is complete, peak averaging 1000 |
| Adhesive-fetch device glues print diaphragm | STR does not detect | STR bit point is complete, peak averaging 1200 | STR bit point is complete, peak averaging 1000 | STR bit point is complete, peak averaging 1200 |
Claims (3)
1. the test kit for high-leveled and difficult contact sample DNA extraction purification, it is characterised in that be made up of Digestive system, adsorption liquid, cleaning mixture 1, cleaning mixture 2, cleaning mixture 3, magnetic bead suspension and eluent seven part;
Wherein, described Digestive system consist of guanidinium isothiocyanate (2.0 ~ 5.0M), guanidine hydrochloride (0.1 ~ 0.5M), TrisHCl(0.02 ~ 0.04M), Tween80(7.0 �� 10-3~1.4��10-2M), EDTA(2.0 �� 10-3~5.0��10-3M), SDS(3.0 �� 10-3~1.5��10-2M);
Described adsorption liquid consists of guanidinium isothiocyanate (2.0 ~ 5.0M), TrisHCl(0.02 ~ 0.04M), ethylene glycol (1.5 ~ 2.5M);
Described cleaning mixture 1 consist of guanidinium isothiocyanate (2.0 ~ 5.0M), TrisHCl(0.02 ~ 0.04M), ethylene glycol (3.0 ~ 4.0M);
Described cleaning mixture 2 consist of dehydrated alcohol: isopropanol: water=7:1:2(volume ratio), add NaCl be 2.0M to concentration;
Described cleaning mixture 3 consist of dehydrated alcohol: isopropanol: water=6:2:2(volume ratio), add NaCl be 0.5M to concentration;
Described magnetic bead suspension is the super-paramagnetism nano magnetic bead of size 100 ~ 2000nm, is dispersed in high purity water, and concentration is 9%(mass percent);
Described eluent is high purity water.
2. the preparation method of a kind of test kit for high-leveled and difficult contact sample DNA extraction purification described in claim 1, comprises the following steps:
Weighing the reagent that preparation Digestive system, adsorption liquid, cleaning mixture 1, cleaning mixture 2, cleaning mixture 3, magnetic bead suspension and eluent relate in proportion respectively, compound method routinely is mix homogeneously respectively.
3. the using method of the test kit for high-leveled and difficult contact sample DNA extraction purification described in claim 1, comprises the following steps:
1) the contact sample taking suitable size is put in centrifuge tube, adds 100 ~ 1000 �� l Digestive systems, adds the E.C. 3.4.21.64 aqueous solution of 10mg/ml according to the ratio of volume ratio 10:1 ~ 20:1; Overturn after mixing, be placed in 56 DEG C of temperature baths 0.5 hour ~ 6 hours;
2) after temperature bath, centrifugal 12000rpm, 3 minutes; Take out supernatant and be placed in new centrifuge tube, in supernatant, add the adsorption liquid of 1 ~ 10 times of supernatant volume, add 5 ~ 30 �� l magnetic bead suspensions; On convertible blending instrument, room temperature mixes absorption 10 ~ 20 minutes continuously;
3) centrifuge tube is placed on magnetic frame and carries out Magneto separate, inhale liquid in pipe abandon, retain magnetic bead;
4) in centrifuge tube, add 150 ~ 400 �� l cleaning mixture 1, cover centrifuge tube lid, put back to magnet stand immediately after vortex vibration mixing, inhale and abandon liquid;
5) in centrifuge tube, add 150 ~ 500 �� l cleaning mixture 2, cover centrifuge tube lid, put back to magnet stand immediately after vortex vibration mixing, inhale and abandon liquid;
6) in centrifuge tube, add 150 ~ 500 �� l cleaning mixture 3, cover centrifuge tube lid, put back to magnet stand immediately after vortex vibration mixing, inhale and abandon liquid, drying at room temperature of uncapping 5 ~ 15 minutes;
7) add 10 ~ 30 �� l eluents, vortex vibration mixing after be put into immediately on magnet stand, treat that magnetic bead is adsorbed onto on centrifuge tube tube wall completely, sucking-off solution be put in another centrifuge tube preserves stand-by.
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| CN108342383A (en) * | 2018-02-28 | 2018-07-31 | 默禾医疗科技（上海）有限公司 | A kind of the rapid extraction kit and its extracting method of high-purity DNA or RNA |
| CN111206029A (en) * | 2019-10-30 | 2020-05-29 | 公安部物证鉴定中心 | Kit and method for extracting DNA from rust pollution trace biological detection material |
| CN111206029B (en) * | 2019-10-30 | 2021-11-12 | 公安部物证鉴定中心 | Kit and method for extracting DNA from rust pollution trace biological detection material |
| CN113604464A (en) * | 2021-07-30 | 2021-11-05 | 中国科学院合肥物质科学研究院 | Kit for extracting and purifying DNA (deoxyribonucleic acid) of trace complex material to be detected and extraction method |
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Application publication date: 20160601 |