CN107058296A - soil urine DNA extraction kit and method - Google Patents
soil urine DNA extraction kit and method Download PDFInfo
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- CN107058296A CN107058296A CN201710391012.5A CN201710391012A CN107058296A CN 107058296 A CN107058296 A CN 107058296A CN 201710391012 A CN201710391012 A CN 201710391012A CN 107058296 A CN107058296 A CN 107058296A
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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
Extracts kit and method are extracted the present invention relates to a kind of soil urine DNA, in the method for the invention, is comprised the following steps：1）DNA is extracted, and is cracked the pedotheque containing urine with soil lysate and Proteinase K, is centrifuged soil and supernatant, supernatant is added and combines liquid, and silicon substrate DNA sorbing materials, separation silicon substrate DNA sorbing materials and liquid are added after mixing, rinse silicon substrate DNA sorbing materials, eluted dna；2）PCR is expanded and electrophoresis, and the DNA that upper step is purified enters performing PCR amplification, and DNA is detected in electrophoresis apparatus；Using this kit and method, absorption of the silica in samples-soil source to DNA is overcome, so as to reduce unnecessary DNA losses, high-quality, high yield soil urine DNA the purpose of acquisition is can reach.
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 DNA theoretical foundation；Under high concentration chaotropic salt and low ph condition, silica can be tied by hydrogen bond and DNA
Close, and the impurity such as protein, lipid, carbohydrate be because can not be combined so as to being removed, after removing chaotropic salt, raising pH value, DNA
Interaction force between silica weakens, and DNA discharges from silica surface, so as to obtain the DNA of purifying；In soil
Containing substantial amounts of silica composition, therefore, it is necessary to consider the titanium dioxide of sample source during to pedotheque progress DNA extractions
Combination of the silicon to DNA, once the silica of DNA and sample source is combined, 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
Survey, two generation sequencing analysis；But, DNA content is relatively low in urine, after urine infiltration soil, by the DNA content of adsorption by soil more
It is low, DNA extractions are carried out for soil urine, tool acquires a certain degree of difficulty.
The content of the invention
The present invention is solves above-mentioned problem there is provided a kind of soil urine DNA extraction kit and method, and it is solved
Certainly the technical scheme of technical problem is：
Soil urine DNA extraction kit, including following reagent：
（a）Soil lysate, its constituent includes surfactant, 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 and the above mixing, the mass concentration of the surfactant is 1-100g/L, surface-active preferably
Agent composition is the mixing of one or both of lauryl sodium sulfate or dodecyl sodium sulfate, surfactant matter preferably
Amount concentration is 1-10g/L；
The chelating agent is one or both of disodium ethylene diamine tetraacetate, ethylenediamine tetra-acetic acid tripotassium, the chaotropic
The concentration of salt is 1-100 mmol/L；
The pH buffer be Tris-HCl, Tris- acetic acid, NaH2PO4-Na2HPO4, KH2PO4-K2HPO4, acetic acid-
Sodium acetate, citric acid-sodium citrate, the pH value of the pH buffer is 7.0-11, and pH buffer preferably is Tris-HCl, excellent
The pH value of the pH buffer of choosing is 7.5-8.5；
（b）With reference to liquid, its constituent includes surfactant, chaotropic salt, pH buffer；
The surfactant is：Triton X-100, TWEEN 20, TWEEN 40, TWEEN 60, TWEEN
80th, the mixing of one or both of NP 40 and the above, the mass concentration of the surfactant is 1-100g/L；
The chaotropic salt is one kind in guanidinium isothiocyanate, guanidine hydrochloride, potassium rhodanide, sodium perchlorate, KI, sodium iodide
Or the mixing of two kinds and the above, the concentration of the chaotropic salt is 3-5 mol/L；
The pH buffer be Tris-HCl, Tris- acetic acid, NaH2PO4-Na2HPO4, KH2PO4-K2HPO4, acetic acid-
Sodium acetate, citric acid-sodium citrate, the pH value of the pH buffer is 4.5-7.0；
（c）Rinsing liquid, the rinsing liquid is 70-80% ethanol；
（d）Eluent, its component is：10 mmol/L Tris-HCl, 0.1 mmol/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
Any one in tunica fibrosa, quartz film, the magnetic nanoparticle of Silica-coated.
Using the kit of the present invention, the method to extract soil urine DNA comprises the following steps：
1）DNA is extracted
The pedotheque containing urine with soil lysate and Proteinase K cracking, centrifuges soil and supernatant, supernatant adds
Enter and combine liquid, silicon substrate DNA sorbing materials, separation silicon substrate DNA sorbing materials and liquid, the DNA absorption of rinsing silicon substrate are added after mixing
Material, eluted dna；Specifically include following steps：
A. the topsoil containing urine is scraped, is dried, 100-200 mg pedotheques are taken, adds in sample cell, then adds
400 ul soil lysates and 20 ul protease k, after concussion is mixed, 75 DEG C of heating are cracked 15 minutes；
B. maximum (top) speed is centrifuged 5 minutes, supernatant is transferred in new sample cell, adds 800 ul combination liquid, is mixed；
C. the mixed liquor of previous step is transferred to silicon substrate DNA sorbing materials, separation liquid and silicon substrate DNA sorbing materials；
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 is expanded and electrophoresis
The DNA that upper step is purified enters performing 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, with reference to liquid, rinsing liquid, wash
De- liquid, using the soil lysate of special composition, before adding and combining liquid, the silica in samples-soil source not with DNA
With reference to centrifuging solid particle and dissolved DNA supernatant, add and be transferred to after combining liquid, mixing containing silicon in supernatant
In base DNA sorbing materials, separation liquid and silicon substrate DNA sorbing materials rinse silicon substrate DNA sorbing materials, and elution is purified
DNA；Compared with prior art, the technology of the present invention overcomes absorption of the silica in samples-soil source to DNA, so as to reduce
Unnecessary DNA losses, can reach high-quality, the high yield soil urine DNA purposes of acquisition.
Brief description of the drawings
Fig. 1 is the DNA extracted in the embodiment of the present invention 1 Multiplex STR amplification collection of illustrative plates.
Fig. 2 is the DNA extracted in the embodiment of the present invention 2 Multiplex STR amplification collection of illustrative plates.
Fig. 3 is the DNA extracted in the embodiment of the present invention 3 Multiplex STR amplification collection of illustrative plates.
Fig. 4 is the DNA extracted in the embodiment of the present invention 4 Multiplex STR amplification collection of illustrative plates.
Embodiment
Below in conjunction with the accompanying drawing of the present invention, technical scheme is clearly and completely described.
Embodiment 1 extracts soil urine DNA with nano SiO 2 particle.
1）DNA is extracted
The topsoil containing urine is scraped, drying takes 100-200 mg pedotheques, adds in sample cell, then add 400
Ul soil lysate and 20 ul protease k, after concussion is mixed, 75 DEG C of heating are cracked 15 minutes；Maximum (top) speed is centrifuged 5 minutes,
Supernatant is transferred in new sample cell, adds 800 ul combination liquid, mixed；Mixed liquor is transferred to equipped with 15 ul titanium dioxides
In the centrifuge tube of nano silicon particles, static 15 minutes after mixing；8000rpm is centrifuged 1 minute and is removed supernatant；Add 500 ul drifts
Washing lotion, is mixed, and 8000rpm is centrifuged 1 minute and removed supernatant；500 ul rinsing liquids are added, are mixed, 8000rpm centrifugations are gone for 1 minute
Except supernatant；70 DEG C are heated 3 minutes；20 ul eluents are added, concussion is mixed, 70 DEG C are heated 3 minutes；8000rpm centrifuges 1 point
Clock, supernatant DNA solution is transferred in new centrifuge tube.
2）PCR is expanded and electrophoresis
PCR amplifications, which are used, contains 4 μ l master mix, 2 μ l in Identifiler Plus kits, 10 μ l amplification systems
Primer set, 4 μ l templates, amplification program is, 95 DEG C, 11 minutes；94 DEG C, 20 seconds, 59 DEG C, 3 minutes, 30 circulations；60
DEG C, 10 minutes；4 DEG C of preservations；Electrophoresis is carried out in ABI 3500XL instruments；Electrophoresis loading system is, 1 μ l PCR primers, 9 μ l
Formamide, wherein adding Liz.
Embodiment 2 extracts soil urine DNA with pellosil
1）DNA is extracted
The topsoil containing urine is scraped, drying takes 100-200 mg pedotheques, adds in sample cell, then add 400
Ul soil lysate and 20 ul protease k, after concussion is mixed, 75 DEG C of heating are cracked 15 minutes；Maximum (top) speed is centrifuged 5 minutes,
Supernatant is transferred in new sample cell, adds 800 ul combination liquid, mixed；Mixed liquor is transferred to the centrifugation equipped with pellosil
In post, 12000rpm is centrifuged 1 minute；Waste liquid in collecting pipe is abandoned, 500 ul rinsing liquids are added, 12000rpm is centrifuged 1 minute；
Waste liquid in collecting pipe is abandoned, 500 ul rinsing liquids are added, 12000rpm is centrifuged 1 minute；Abandon waste liquid in collecting pipe, 12000rpm from
The heart 3 minutes, centrifugal column is transferred in new centrifuge tube, and 70 DEG C are heated 3 minutes；20 ul eluents are added, 70 DEG C are heated 3 points
Clock；12000rpm is centrifuged 1 minute, and it is DNA solution to abandon liquid in centrifugal column, centrifuge tube.
2）PCR is expanded and electrophoresis
PCR amplifications, which are used, contains 4 μ l master mix, 2 μ l in Identifiler Plus kits, 10 μ l amplification systems
Primer set, 4 μ l templates, amplification program is, 95 DEG C, 11 minutes；94 DEG C, 20 seconds, 59 DEG C, 3 minutes, 30 circulations；60
DEG C, 10 minutes；4 DEG C of preservations；Electrophoresis is carried out in ABI 3500XL instruments；Electrophoresis loading system is, 1 μ l PCR primers, 9 μ l
Formamide, wherein adding Liz.
Embodiment 3 extracts soil urine DNA with glass fibre membrane
1）DNA is extracted
The topsoil containing urine is scraped, drying takes 100-200 mg pedotheques, adds in sample cell, then add 400
Ul soil lysate and 20 ul protease k, after concussion is mixed, 75 DEG C of heating are cracked 15 minutes；Maximum (top) speed is centrifuged 5 minutes,
Supernatant is transferred in new sample cell, adds 800 ul combination liquid, mixed；Mixed liquor be transferred to equipped with glass fibre from
In stem, 12000rpm is centrifuged 1 minute；Waste liquid in collecting pipe is abandoned, 500 ul rinsing liquids are added, 12000rpm is centrifuged 1 minute；
Waste liquid in collecting pipe is abandoned, 500 ul rinsing liquids are added, 12000rpm is centrifuged 1 minute；Abandon waste liquid in collecting pipe, 12000rpm
Centrifugation 3 minutes, centrifugal column is transferred in new centrifuge tube, and 70 DEG C are heated 3 minutes；Add 20 ul eluents, 70 DEG C of heating 3
Minute；12000rpm is centrifuged 1 minute, and it is DNA solution to abandon liquid in centrifugal column, centrifuge tube.
2）PCR is expanded and electrophoresis
PCR amplifications, which are used, contains 4 μ l master mix, 2 μ l in Identifiler Plus kits, 10 μ l amplification systems
Primer set, 4 μ l templates, amplification program is, 95 DEG C, 11 minutes；94 DEG C, 20 seconds, 59 DEG C, 3 minutes, 30 circulations；60
DEG C, 10 minutes；4 DEG C of preservations；Electrophoresis is carried out in ABI 3500XL instruments；Electrophoresis loading system is, 1 μ l PCR primers, 9 μ l
Formamide, wherein adding Liz.
Embodiment 4 extracts soil urine DNA with quartz film
1）DNA is extracted
The topsoil containing urine is scraped, drying takes 100-200 mg pedotheques, adds in sample cell, then add 400
Ul soil lysate and 20 ul protease k, after concussion is mixed, 75 DEG C of heating are cracked 15 minutes；Maximum (top) speed is centrifuged 5 minutes,
Supernatant is transferred in new sample cell, adds 800 ul combination liquid, mixed；Mixed liquor is transferred to the centrifugation equipped with quartz film
In post, 12000rpm is centrifuged 1 minute；Waste liquid in collecting pipe is abandoned, 500 ul rinsing liquids are added, 12000rpm is centrifuged 1 minute；
Waste liquid in collecting pipe is abandoned, 500 ul rinsing liquids are added, 12000rpm is centrifuged 1 minute；Abandon waste liquid in collecting pipe, 12000rpm from
The heart 3 minutes, centrifugal column is transferred in new centrifuge tube, and 70 DEG C are heated 3 minutes；20 ul eluents are added, 70 DEG C are heated 3 points
Clock；12000rpm is centrifuged 1 minute, and it is DNA solution to abandon liquid in centrifugal column, centrifuge tube.
2）PCR is expanded and electrophoresis
PCR amplifications, which are used, contains 4 μ l master mix, 2 μ l in Identifiler Plus kits, 10 μ l amplification systems
Primer set, 4 μ l templates, amplification program is, 95 DEG C, 11 minutes；94 DEG C, 20 seconds, 59 DEG C, 3 minutes, 30 circulations；60
DEG C, 10 minutes；4 DEG C of preservations；Electrophoresis is carried out in ABI 3500XL instruments；Electrophoresis loading system is, 1 μ l PCR primers, 9 μ l
Formamide, wherein adding Liz.
Embodiment 5 extracts soil urine DNA with the magnetic nanoparticle of Silica-coated
1）DNA is extracted
The topsoil containing urine is scraped, drying takes 100-200 mg pedotheques, adds in sample cell, then add 400
Ul soil lysate and 20 ul protease k, after concussion is mixed, 75 DEG C of heating are cracked 15 minutes；Maximum (top) speed is centrifuged 5 minutes,
Supernatant is transferred in new sample cell, adds 800 ul combination liquid, mixed；Mixed liquor is transferred to equipped with 15 ul titanium dioxides
In the centrifuge tube of the magnetic nanoparticle of silicon parcel, static 15 minutes after mixing；Upper magnetic frame is adsorbed 2 minutes, and supernatant is abandoned in suction；
500 ul rinsing liquids are added, are mixed, upper magnetic frame is adsorbed 1 minute, supernatant is abandoned in suction；500 ul rinsing liquids are added, are mixed, upper magnetic force
Frame is adsorbed 1 minute, and supernatant is abandoned in suction；70 DEG C are heated 3 minutes；20 ul eluents are added, concussion is mixed, 70 DEG C are heated 3 minutes；On
Magnetic frame is adsorbed 1 minute, and supernatant DNA solution is transferred in new centrifuge tube.
2）PCR is expanded and electrophoresis
PCR amplifications, which are used, contains 4 μ l master mix, 2 μ l in Identifiler Plus kits, 10 μ l amplification systems
Primer set, 4 μ l templates, amplification program is, 95 DEG C, 11 minutes；94 DEG C, 20 seconds, 59 DEG C, 3 minutes, 30 circulations；60
DEG C, 10 minutes；4 DEG C of preservations；Electrophoresis is carried out in ABI 3500XL instruments；Electrophoresis loading system is, 1 μ l PCR primers, 9 μ l
Formamide, wherein adding Liz.
The embodiment of the present invention is the foregoing is only, is not intended to limit the invention, for the technology of this area
For personnel, the present invention can have various modifications and variations；Any modification for being made within the spirit and principles of the invention,
Equivalent substitution, improvement etc., should be included in the scope of the protection.
Claims (2)
1. soil urine DNA extraction kit, it is characterised in that including following reagent：
（a）Soil lysate, its constituent includes 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 mixing of the one or two kinds of and above, the mass concentration of the surfactant is 1-100g/L, preferred surfactant into
Point it is the mixing of one or both of lauryl sodium sulfate or dodecyl sodium sulfate, surfactant qualities preferably are dense
Spend for 1-10g/L；
The chelating agent is one or both of disodium ethylene diamine tetraacetate, ethylenediamine tetra-acetic acid tripotassium, the chaotropic salt
Concentration is 1-100 mmol/L；
The pH buffer is Tris-HCl, Tris- acetic acid, NaH2PO4-Na2HPO4, KH2PO4-K2HPO4, acetic acid-acetic acid
Sodium, citric acid-sodium citrate, the pH value of the pH buffer is 7.0-11, and pH buffer preferably is Tris-HCl, preferably
The pH value of pH buffer is 7.5-8.5；
（b）With reference to liquid, its constituent includes surfactant, chaotropic salt, pH buffer；
The surfactant is：Triton X-100, TWEEN 20, TWEEN 40, TWEEN 60, TWEEN 80,
One or both of NP 40 and the above mixing, the mass concentration of the surfactant is 1-100g/L；
The chaotropic salt is one kind or two in guanidinium isothiocyanate, guanidine hydrochloride, potassium rhodanide, sodium perchlorate, KI, sodium iodide
The mixing of kind and the above, the concentration of the chaotropic salt is 3-5 mol/L；
The pH buffer is Tris-HCl, Tris- acetic acid, NaH2PO4-Na2HPO4, KH2PO4-K2HPO4, acetic acid-acetic acid
Sodium, citric acid-sodium citrate, the pH value of the pH buffer is 4.5-7.0；
（c）Rinsing liquid, the rinsing liquid is 70-80% ethanol；
（d）Eluent, its component is：10 mmol/L Tris-HCl, 0.1 mmol/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
Any one in film, quartz film, the magnetic nanoparticle of Silica-coated.
2. the method that the kit described in usage right requirement 1 extracts soil urine DNA, it is characterised in that comprise the following steps：
1）DNA is extracted
The pedotheque containing urine with soil lysate and Proteinase K cracking, centrifuges soil and supernatant, supernatant adds
Enter and combine liquid, silicon substrate DNA sorbing materials, separation silicon substrate DNA sorbing materials and liquid, the DNA absorption of rinsing silicon substrate are added after mixing
Material, eluted dna；Specifically include following steps：
（1）The topsoil containing urine is scraped, drying takes 100-200 mg pedotheques, adds in sample cell, then add
400 ul soil lysates and 20 ul protease k, after concussion is mixed, 75 DEG C of heating are cracked 15 minutes；
（2）Maximum (top) speed is centrifuged 5 minutes, supernatant is transferred in new sample cell, adds 800 ul combination liquid, is mixed；
（3）The mixed liquor of previous step is transferred to silicon substrate DNA sorbing materials, separation liquid and silicon substrate DNA sorbing materials；
（4）Silicon substrate DNA sorbing materials are rinsed with rinsing liquid；
（5）With the DNA in elution silicon-based adsorption material material；
2）PCR is expanded and electrophoresis
The DNA that upper step is purified enters performing PCR amplification, and DNA is detected in electrophoresis apparatus.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710391012.5A CN107058296B (en) | 2017-05-27 | 2017-05-27 | soil urine DNA extraction kit and method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710391012.5A CN107058296B (en) | 2017-05-27 | 2017-05-27 | soil urine DNA extraction kit and method |
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| CN107058296A true CN107058296A (en) | 2017-08-18 |
| CN107058296B CN107058296B (en) | 2018-09-25 |
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| CN201710391012.5A Expired - Fee Related CN107058296B (en) | 2017-05-27 | 2017-05-27 | soil urine DNA extraction kit and method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| WO2020140975A1 (en) * | 2019-01-03 | 2020-07-09 | Hangzhou New Horizon Health Technology Co. Ltd. | Compositions and methods for urine sample storage and dna extraction |
| 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 |
| CN113549615A (en) * | 2021-08-30 | 2021-10-26 | 浙江大学 | Method for separating and extracting high-quality strawberry genome DNA |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020140975A1 (en) * | 2019-01-03 | 2020-07-09 | Hangzhou New Horizon Health Technology Co. Ltd. | Compositions and methods for urine sample storage and dna extraction |
| CN111902545A (en) * | 2019-01-03 | 2020-11-06 | 杭州诺辉健康科技有限公司 | Composition and method for urine sample preservation and DNA extraction |
| EP3906319A4 (en) * | 2019-01-03 | 2022-11-16 | 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 |
| CN113549615A (en) * | 2021-08-30 | 2021-10-26 | 浙江大学 | Method for separating and extracting high-quality strawberry genome DNA |
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| Publication number | Publication date |
|---|---|
| CN107058296B (en) | 2018-09-25 |
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