CN107058296B - soil urine DNA extraction kit and method - Google Patents
soil urine DNA extraction kit and method Download PDFInfo
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- CN107058296B CN107058296B CN201710391012.5A CN201710391012A CN107058296B CN 107058296 B CN107058296 B CN 107058296B CN 201710391012 A CN201710391012 A CN 201710391012A CN 107058296 B CN107058296 B CN 107058296B
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- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
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Abstract
The present invention relates to a kind of soil urine DNA extraction extracts kits and method to include the following steps in the method for the invention：1）DNA is extracted, and is cracked the pedotheque containing urine with soil lysate and Proteinase K, is centrifuged soil and supernatant, supernatant, which is added, combines liquid, and silicon substrate DNA sorbing materials are added after mixing, detach silicon substrate DNA sorbing materials and liquid, rinse silicon substrate DNA sorbing materials, eluted dna；2）PCR amplification and electrophoresis, the DNA that upper step is purified carry out PCR amplification, DNA are detected in electrophoresis apparatus；Using this kit and method, the absorption of the silica to DNA in samples-soil source is overcome, to reduce unnecessary DNA losses, can reach and obtain high quality, the purpose of high yield soil urine DNA.
Description
Technical field
The invention belongs to nucleic acid extraction technical field of purification, and in particular to a kind of soil urine DNA extraction kit and side
Method.
Background technology
Silica can be reversible under the conditions of salt and certain ph combination DNA, this is also silica bead method, paramagnetic particle method, silica gel
Embrane method extracts the theoretical foundation of DNA；Under high concentration chaotropic salt and low ph condition, silica can be tied by hydrogen bond and DNA
It closes, and the impurity such as protein, lipid, carbohydrate are removed because that cannot be combined, after removal chaotropic salt, raising pH value, DNA
Interaction force between silica weakens, and DNA is discharged from silica surface, to obtain the DNA of purifying；In soil
Containing a large amount of silica composition, therefore, when carrying out DNA extractions to pedotheque, the titanium dioxide for considering sample source is needed
Combination of the silicon to DNA, once the silica of DNA and sample source combines, DNA will be with soil solid particulate matter
It is removed together.
Contain a certain amount of DNA in urine, DNA can be extracted by a variety of methods, and for PCR detections, the inspection of STR partings
It surveys, two generation sequencing analysis；But DNA content is relatively low in urine, after urine infiltrates soil, more by the DNA content of adsorption by soil
It is low, it to be directed to soil urine and carry out DNA extractions, tool acquires a certain degree of difficulty.
Invention content
The present invention is solves the problems, such as above-mentioned, provides a kind of soil urine DNA extraction kit and method, solves
Certainly the technical solution of technical problem is：
Soil urine DNA extraction kit, including following reagent：(a) soil lysate, constituent include that surface is lived
Property agent, chelating agent, pH buffer；
The surfactant is：Lauryl sodium sulfate, dodecyl sodium sulfate, sodium N-lauroyl sarcosinate, poly- second
Glycol octyl phenyl ether, TWEEN 20, TWEEN 40, TWEEN 60, TWEEN 80, NP 40, cetyl trimethylammonium bromide
One or both of or more mixing, the mass concentration of the surfactant is 1-100g/L, preferred surface-active
Agent ingredient is the mixing of one or both of lauryl sodium sulfate or dodecyl sodium sulfate, preferred surfactant matter
Measure a concentration of 1-10g/L；
The chelating agent is one or both of disodium ethylene diamine tetraacetate, ethylenediamine tetra-acetic acid tripotassium, the chelating
A concentration of 1-100mmol/L of agent；
The pH buffer is Tris-HCl, Tris- acetic acid, NaH2PO4-Na2HPO4, KH2PO4-K2HPO4, acetic acid-
The pH value of sodium acetate, citric acid-sodium citrate, the pH buffer is 7.0-11, and preferred pH buffer is Tris-HCl, excellent
The pH value of the pH buffer of choosing is 7.5-8.5；(b) it includes surfactant, chaotropic salt, pH bufferings to combine liquid, constituent
Agent；
The surfactant is：Triton X-100, TWEEN 20, TWEEN 40, TWEEN 60, TWEEN
80, the mass concentration of the mixing of one or both of NP 40 or more, the surfactant is 1-100g/L；
The chaotropic salt is one kind in guanidinium isothiocyanate, guanidine hydrochloride, potassium rhodanide, sodium perchlorate, potassium iodide, sodium iodide
Or two kinds or more of mixing, a concentration of 3-5mol/L of the chaotropic salt；
The pH buffer is Tris-HCl, Tris- acetic acid, NaH2PO4-Na2HPO4, KH2PO4-K2HPO4, acetic acid-
The pH value of sodium acetate, citric acid-sodium citrate, the pH buffer is 4.5-7.0；
(c) rinsing liquid, the rinsing liquid are 70-80% ethyl alcohol；
(d) eluent, group are divided into：10mmol/L Tris-HCl, 0.1mmol/L disodium ethylene diamine tetraacetates, pH
8.0；
(e) silicon substrate DNA sorbing materials, the silicon substrate DNA sorbing materials are nano SiO 2 particle, pellosil, glass
Tunica fibrosa, quartz film, Silica-coated magnetic nanoparticle in any one.
Using the kit of the present invention, the method to extract soil urine DNA includes the following steps：
1) DNA extractions soil lysate and Proteinase K crack the pedotheque containing urine, centrifuge soil and upper
Clear liquid, supernatant, which is added, combines liquid, and silicon substrate DNA sorbing materials are added after mixing, detaches silicon substrate DNA sorbing materials and liquid, drift
Wash silicon substrate DNA sorbing materials, eluted dna；Specifically include following steps：A. the topsoil containing urine is scraped, dries, takes
100-200mg pedotheques are added in sample cell, and 400ul soil lysate and 20ul protease k is then added, and shake mixing
Afterwards, 75 DEG C of heating crack 15 minutes；B. maximum (top) speed centrifuges 5 minutes, supernatant is transferred in new sample cell, is added
800ul combination liquid, mixing；C. the mixed liquor of previous step is transferred to silicon substrate DNA sorbing materials, detaches liquid and silicon substrate DNA inhales
Enclosure material；D. silicon substrate DNA sorbing materials are rinsed with rinsing liquid；E. the DNA in elution silicon-based adsorption material material is used；2)PCR
Amplification and electrophoresis
The DNA that upper step is purified carries out PCR amplification, and DNA is detected in electrophoresis apparatus.The invention has the advantages that：
The present invention soil urine DNA extraction kit and method, prepare soil lysate, in conjunction with liquid, rinsing liquid, wash
De- liquid, using the soil lysate of special composition, before being added and combining liquid, the silica in samples-soil source not with DNA
In conjunction with centrifuging solid particle and dissolved the supernatant of DNA, be added in supernatant and combine liquid, be transferred to containing silicon after mixing
In base DNA sorbing materials, liquid and silicon substrate DNA sorbing materials are detached, rinses silicon substrate DNA sorbing materials, elution is purified
DNA；Compared with prior art, the technology of the present invention overcomes the absorption of the silica to DNA in samples-soil source, to reduce
Unnecessary DNA losses, can reach and obtain high quality, high yield soil urine DNA purposes.
Description of the drawings
Fig. 1 is the Multiplex STR amplification collection of illustrative plates of the DNA extracted in the embodiment of the present invention 1.
Fig. 2 is the Multiplex STR amplification collection of illustrative plates of the DNA extracted in the embodiment of the present invention 2.
Fig. 3 is the Multiplex STR amplification collection of illustrative plates of the DNA extracted in the embodiment of the present invention 3.
Fig. 4 is the Multiplex STR amplification collection of illustrative plates of the DNA extracted in the embodiment of the present invention 4.
Specific implementation mode
Below in conjunction with the attached drawing of the present invention, technical scheme of the present invention is clearly and completely described.
Embodiment 1 is with nano SiO 2 particle extraction soil urine DNA.
1) topsoil of the DNA extractions scraping containing urine, drying take 100-200mg pedotheques, and sample cell is added
In, 400ul soil lysate and 20ul protease k is then added, after shaking mixing, 75 DEG C of heating crack 15 minutes；Maximum turns
Speed centrifugation 5 minutes, supernatant is transferred in new sample cell, and 800ul combination liquid, mixing is added；Mixed liquor, which is transferred to, to be equipped with
In the centrifuge tube of 15ul nano SiO 2 particles, static 15 minutes after mixing；8000rpm centrifuges 1 minute removal supernatant；Add
Enter 500ul rinsing liquids, mixing, 8000rpm centrifuges 1 minute removal supernatant；Be added 500ul rinsing liquids, mixing, 8000rpm from
1 minute removal supernatant of the heart；70 DEG C are heated 3 minutes；20ul eluents are added, shake mixing, 70 DEG C are heated 3 minutes；8000rpm
Centrifugation 1 minute, supernatant DNA solution is transferred in new centrifuge tube.
2) PCR amplification and electrophoresis PCR amplification use Identifiler Plus kits, contain 4 μ l in 10 μ l amplification systems
Master mix, 2 μ l primer set, 4 μ l templates, amplification program is 95 DEG C, 11 minutes；94 DEG C, 20 seconds, 59 DEG C, 3 points
Clock, 30 cycles；60 DEG C, 10 minutes；4 DEG C of preservations；Electrophoresis carries out in ABI 3500XL instruments；Electrophoresis loading system is 1 μ l
PCR product, 9 μ l formamides, wherein having added Liz.
Embodiment 2 is with pellosil extraction soil urine DNA
1) topsoil of the DNA extractions scraping containing urine, drying take 100-200mg pedotheques, and sample cell is added
In, 400ul soil lysate and 20ul protease k is then added, after shaking mixing, 75 DEG C of heating crack 15 minutes；Maximum turns
Speed centrifugation 5 minutes, supernatant is transferred in new sample cell, and 800ul combination liquid, mixing is added；Mixed liquor, which is transferred to, to be equipped with
In the centrifugal column of pellosil, 12000rpm is centrifuged 1 minute；Waste liquid in collecting pipe is abandoned, 500ul rinsing liquids, 12000rpm is added
Centrifugation 1 minute；Waste liquid in collecting pipe is abandoned, 500ul rinsing liquids are added, 12000rpm is centrifuged 1 minute；It abandons in collecting pipe and gives up
Liquid, 12000rpm are centrifuged 3 minutes, centrifugal column are transferred in new centrifuge tube, and 70 DEG C are heated 3 minutes；20ul eluents are added,
70 DEG C are heated 3 minutes；12000rpm is centrifuged 1 minute, abandons centrifugal column, and liquid is DNA solution in centrifuge tube.
2) PCR amplification and electrophoresis PCR amplification use Identifiler Plus kits, contain 4 μ l in 10 μ l amplification systems
Master mix, 2 μ l primer set, 4 μ l templates, amplification program is 95 DEG C, 11 minutes；94 DEG C, 20 seconds, 59 DEG C, 3 points
Clock, 30 cycles；60 DEG C, 10 minutes；4 DEG C of preservations；Electrophoresis carries out in ABI 3500XL instruments；Electrophoresis loading system is 1 μ l
PCR product, 9 μ l formamides, wherein having added Liz.
Embodiment 3 is with glass fibre membrane extraction soil urine DNA
1) topsoil of the DNA extractions scraping containing urine, drying take 100-200mg pedotheques, and sample cell is added
In, 400ul soil lysate and 20ul protease k is then added, after shaking mixing, 75 DEG C of heating crack 15 minutes；Maximum turns
Speed centrifugation 5 minutes, supernatant is transferred in new sample cell, and 800ul combination liquid, mixing is added；Mixed liquor, which is transferred to, to be equipped with
In the centrifugal column of glass fibre, 12000rpm is centrifuged 1 minute；Waste liquid in collecting pipe is abandoned, 500ul rinsing liquids are added,
12000rpm is centrifuged 1 minute；Waste liquid in collecting pipe is abandoned, 500ul rinsing liquids are added, 12000rpm is centrifuged 1 minute；Abandon collection
Waste liquid in pipe, 12000rpm are centrifuged 3 minutes, centrifugal column are transferred in new centrifuge tube, and 70 DEG C are heated 3 minutes；20ul is added
Eluent, 70 DEG C are heated 3 minutes；12000rpm is centrifuged 1 minute, abandons centrifugal column, and liquid is DNA solution in centrifuge tube.
2) PCR amplification and electrophoresis PCR amplification use Identifiler Plus kits, contain 4 μ l in 10 μ l amplification systems
Master mix, 2 μ l primer set, 4 μ l templates, amplification program is 95 DEG C, 11 minutes；94 DEG C, 20 seconds, 59 DEG C, 3 points
Clock, 30 cycles；60 DEG C, 10 minutes；4 DEG C of preservations；Electrophoresis carries out in ABI 3500XL instruments；Electrophoresis loading system is 1 μ l
PCR product, 9 μ l formamides, wherein having added Liz.
Embodiment 4 is with quartz film extraction soil urine DNA
1) topsoil of the DNA extractions scraping containing urine, drying take 100-200mg pedotheques, and sample cell is added
In, 400ul soil lysate and 20ul protease k is then added, after shaking mixing, 75 DEG C of heating crack 15 minutes；Maximum turns
Speed centrifugation 5 minutes, supernatant is transferred in new sample cell, and 800ul combination liquid, mixing is added；Mixed liquor, which is transferred to, to be equipped with
In the centrifugal column of quartz film, 12000rpm is centrifuged 1 minute；Waste liquid in collecting pipe is abandoned, 500ul rinsing liquids, 12000rpm is added
Centrifugation 1 minute；Waste liquid in collecting pipe is abandoned, 500ul rinsing liquids are added, 12000rpm is centrifuged 1 minute；It abandons in collecting pipe and gives up
Liquid, 12000rpm are centrifuged 3 minutes, centrifugal column are transferred in new centrifuge tube, and 70 DEG C are heated 3 minutes；20ul eluents are added,
70 DEG C are heated 3 minutes；12000rpm is centrifuged 1 minute, abandons centrifugal column, and liquid is DNA solution in centrifuge tube.
2) PCR amplification and electrophoresis PCR amplification use Identifiler Plus kits, contain 4 μ l in 10 μ l amplification systems
Master mix, 2 μ l primer set, 4 μ l templates, amplification program is 95 DEG C, 11 minutes；94 DEG C, 20 seconds, 59 DEG C, 3 points
Clock, 30 cycles；60 DEG C, 10 minutes；4 DEG C of preservations；Electrophoresis carries out in ABI 3500XL instruments；Electrophoresis loading system is 1 μ l
PCR product, 9 μ l formamides, wherein having added Liz.
Embodiment 5 is with the magnetic nanoparticle extraction soil urine DNA of Silica-coated
1) topsoil of the DNA extractions scraping containing urine, drying take 100-200mg pedotheques, and sample cell is added
In, 400ul soil lysate and 20ul protease k is then added, after shaking mixing, 75 DEG C of heating crack 15 minutes；Maximum turns
Speed centrifugation 5 minutes, supernatant is transferred in new sample cell, and 800ul combination liquid, mixing is added；Mixed liquor, which is transferred to, to be equipped with
In the centrifuge tube of the magnetic nanoparticle of 15ul Silica-coateds, static 15 minutes after mixing；Upper magnetic frame adsorbs 2 minutes,
Supernatant is abandoned in suction；500ul rinsing liquids, mixing is added, upper magnetic frame adsorbs 1 minute, and supernatant is abandoned in suction；500ul rinsing liquids are added, mix
Even, upper magnetic frame adsorbs 1 minute, and supernatant is abandoned in suction；70 DEG C are heated 3 minutes；20ul eluents are added, shake mixing, 70 DEG C of heating 3
Minute；Upper magnetic frame adsorbs 1 minute, and supernatant DNA solution is transferred in new centrifuge tube.
2) PCR amplification and electrophoresis PCR amplification use Identifiler Plus kits, contain 4 μ l in 10 μ l amplification systems
Master mix, 2 μ l primer set, 4 μ l templates, amplification program is 95 DEG C, 11 minutes；94 DEG C, 20 seconds, 59 DEG C, 3 points
Clock, 30 cycles；60 DEG C, 10 minutes；4 DEG C of preservations；Electrophoresis carries out in ABI 3500XL instruments；Electrophoresis loading system is 1 μ l
PCR product, 9 μ l formamides, wherein having added Liz.The foregoing is merely the specific implementation modes of the present invention, are not used to limit
The system present invention, for those skilled in the art, the invention may be variously modified and varied；All spirit in the present invention
With any modification, equivalent replacement, improvement and so within principle, should all be included in the protection scope of the present invention.
Claims (3)
1. soil urine DNA extraction kit, which is characterized in that including following reagent：
(a) soil lysate, constituent include surfactant, chelating agent, pH buffer；
The surfactant is：Lauryl sodium sulfate, dodecyl sodium sulfate, sodium N-lauroyl sarcosinate, polyethylene glycol
In octyl phenyl ether, TWEEN 20, TWEEN 40, TWEEN 60, TWEEN 80, NP 40, cetyl trimethylammonium bromide
The mass concentration of one or two kinds of or more mixing, the surfactant is 1-100g/L；
The chelating agent is disodium ethylene diamine tetraacetate, one or both of ethylenediamine tetra-acetic acid tripotassium, the chelating agent
A concentration of 1-100mmol/L；
The pH buffer is Tris-HCl, Tris- acetic acid, NaH2PO4-Na2HPO4, KH2PO4-K2HPO4, acetic acid-acetic acid
The pH value of sodium, citric acid-sodium citrate, the pH buffer is 7.0-11；
(b) liquid is combined, constituent includes surfactant, chaotropic salt, pH buffer；
The surfactant is：Triton X-100, TWEEN 20, TWEEN 40, TWEEN 60, TWEEN 80,
The mass concentration of the mixing of one or both of NP 40 or more, the surfactant is 1-100g/L；
The chaotropic salt is guanidinium isothiocyanate, guanidine hydrochloride, potassium rhodanide, sodium perchlorate, potassium iodide, one kind in sodium iodide or two
Kind or more mixing, a concentration of 3-5mol/L of the chaotropic salt；
The pH buffer is Tris-HCl, Tris- acetic acid, NaH2PO4-Na2HPO4, KH2PO4-K2HPO4, acetic acid-acetic acid
The pH value of sodium, citric acid-sodium citrate, the pH buffer is 4.5-7.0；
(c) rinsing liquid, the rinsing liquid are 70-80% ethyl alcohol；
(d) eluent, group are divided into：10mmol/L Tris-HCl, 0.1mmol/L disodium ethylene diamine tetraacetates, pH 8.0；
(e) silicon substrate DNA sorbing materials, the silicon substrate DNA sorbing materials are nano SiO 2 particle, pellosil, glass fibre
Film, quartz film, Silica-coated magnetic nanoparticle in any one.
2. soil urine DNA extraction kit as described in claim 1, which is characterized in that the table in soil lysate
Face activating agent is the mixing of one or both of lauryl sodium sulfate or dodecyl sodium sulfate, the surfactant matter
Measure a concentration of 1-10g/L；
The pH buffer in soil lysate is Tris-HCl, and the pH value of pH buffer is 7.5-8.5.
3. using the method for the kit extraction soil urine DNA described in claims 1 or 22, which is characterized in that including following step
Suddenly：
1) DNA is extracted
The pedotheque containing urine is cracked with soil lysate and Proteinase K, soil is centrifuged and supernatant, supernatant adds
Enter to combine liquid, silicon substrate DNA sorbing materials are added after mixing, detaches silicon substrate DNA sorbing materials and liquid, the DNA absorption of rinsing silicon substrate
Material, eluted dna；Specifically include following steps：(1) topsoil containing urine is scraped, drying takes 100-200mg soil
Sample is added in sample cell, and 400ul soil lysate and 20ul protease k is then added, and after shaking mixing, 75 DEG C of heating are split
Solution 15 minutes；
(2) maximum (top) speed centrifuges 5 minutes, supernatant is transferred in new sample cell, and 800ul combination liquid, mixing is added；
(3) mixed liquor of previous step is transferred to silicon substrate DNA sorbing materials, detaches liquid and silicon substrate DNA sorbing materials；
(4) silicon substrate DNA sorbing materials are rinsed with rinsing liquid；
(5) DNA in elution silicon-based adsorption material material is used；
2) PCR amplification and electrophoresis
The DNA that upper step is purified carries out PCR amplification, and DNA is detected in electrophoresis apparatus.
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| CN201710391012.5A CN107058296B (en) | 2017-05-27 | 2017-05-27 | soil urine DNA extraction kit and method |
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| CN201710391012.5A CN107058296B (en) | 2017-05-27 | 2017-05-27 | soil urine DNA extraction kit and method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CA3119928A1 (en) * | 2019-01-03 | 2020-07-09 | Hangzhou New Horizon Health Technology Co. Ltd. | Compositions and methods for urine sample storage and dna extraction |
| CN110656026A (en) * | 2019-09-29 | 2020-01-07 | 成都市第五人民医院 | Automatic urine DNA purification device |
| CN111218386A (en) * | 2020-03-25 | 2020-06-02 | 广州高盛生物科技股份有限公司 | Silica bead method nucleic acid extraction equipment and use method and application thereof |
| CN112708618A (en) * | 2020-12-30 | 2021-04-27 | 广州博江生物科技有限公司 | Lysate for quickly lysing cells, viruses and bacteria to extract nucleic acid |
| CN113549615B (en) * | 2021-08-30 | 2023-07-25 | 浙江大学 | Method for separating and extracting high-quality strawberry genome DNA |
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| CN100410377C (en) * | 2006-08-29 | 2008-08-13 | 中国科学院南海海洋研究所 | Kit for extracting microbial genome DNA from soil and its method |
| CN105441423A (en) * | 2015-12-03 | 2016-03-30 | 安徽远大机械制造有限公司 | Method for rapidly extracting microbial genomes from soil sample |
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Effective date of registration: 20191216 Address after: 8316, floor 3, R & D building, No. 5, Gushui Road, Changfeng Shuangfeng Economic Development Zone, Hefei City, Anhui Province, 230000 Patentee after: Anhui senpeng Biotechnology Co., Ltd Address before: Heyang city of Rongcheng West Weihai 264300 Shandong Province Patentee before: Shandong Peng Sen Biotechnology Co. Ltd. |
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