CN106282160B - Lysate, extracting solution, cracking and extracting method, kit, application and PCR system - Google Patents
Lysate, extracting solution, cracking and extracting method, kit, application and PCR system Download PDFInfo
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- CN106282160B CN106282160B CN201610656613.XA CN201610656613A CN106282160B CN 106282160 B CN106282160 B CN 106282160B CN 201610656613 A CN201610656613 A CN 201610656613A CN 106282160 B CN106282160 B CN 106282160B
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- lysate
- neutralizer
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- 238000000034 method Methods 0.000 title claims abstract description 75
- 239000006166 lysate Substances 0.000 title claims description 78
- 238000005336 cracking Methods 0.000 title claims description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 41
- 238000007400 DNA extraction Methods 0.000 claims abstract description 27
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims abstract description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 25
- 229960001484 edetic acid Drugs 0.000 claims abstract description 23
- 239000011780 sodium chloride Substances 0.000 claims abstract description 20
- -1 hydroxide ions Chemical class 0.000 claims abstract 2
- 239000000243 solution Substances 0.000 claims description 137
- 210000001519 tissue Anatomy 0.000 claims description 130
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 21
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 19
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 19
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 19
- RCEAADKTGXTDOA-UHFFFAOYSA-N OS(O)(=O)=O.CCCCCCCCCCCC[Na] Chemical compound OS(O)(=O)=O.CCCCCCCCCCCC[Na] RCEAADKTGXTDOA-UHFFFAOYSA-N 0.000 claims description 17
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 17
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 claims description 16
- 210000000988 bone and bone Anatomy 0.000 claims description 16
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 claims description 16
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- 239000000872 buffer Substances 0.000 claims description 11
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- 239000007983 Tris buffer Substances 0.000 claims description 8
- 229940098773 bovine serum albumin Drugs 0.000 claims description 8
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- 230000009467 reduction Effects 0.000 abstract description 2
- 230000009089 cytolysis Effects 0.000 abstract 5
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 abstract 2
- 230000002934 lysing effect Effects 0.000 abstract 1
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- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 12
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- 241000196324 Embryophyta Species 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 108020005196 Mitochondrial DNA Proteins 0.000 description 2
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- JGBUYEVOKHLFID-UHFFFAOYSA-N gelred Chemical compound [I-].[I-].C=1C(N)=CC=C(C2=CC=C(N)C=C2[N+]=2CCCCCC(=O)NCCCOCCOCCOCCCNC(=O)CCCCC[N+]=3C4=CC(N)=CC=C4C4=CC=C(N)C=C4C=3C=3C=CC=CC=3)C=1C=2C1=CC=CC=C1 JGBUYEVOKHLFID-UHFFFAOYSA-N 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 229920000136 polysorbate Polymers 0.000 description 2
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- 239000011734 sodium Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 150000005846 sugar alcohols Chemical class 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
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- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- BDOYKFSQFYNPKF-UHFFFAOYSA-N 2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid;sodium Chemical compound [Na].[Na].OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O BDOYKFSQFYNPKF-UHFFFAOYSA-N 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
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- 241000283690 Bos taurus Species 0.000 description 1
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- 241000282988 Capreolus Species 0.000 description 1
- 241000282983 Capreolus capreolus Species 0.000 description 1
- 241000282985 Cervus Species 0.000 description 1
- 241000283026 Cervus elaphus Species 0.000 description 1
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- 241001596950 Larimichthys crocea Species 0.000 description 1
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
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- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
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- 239000007853 buffer solution Substances 0.000 description 1
- AIYUHDOJVYHVIT-UHFFFAOYSA-M caesium chloride Chemical compound [Cl-].[Cs+] AIYUHDOJVYHVIT-UHFFFAOYSA-M 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
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- 238000012360 testing method Methods 0.000 description 1
- UEUXEKPTXMALOB-UHFFFAOYSA-J tetrasodium;2-[2-[bis(carboxylatomethyl)amino]ethyl-(carboxylatomethyl)amino]acetate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]C(=O)CN(CC([O-])=O)CCN(CC([O-])=O)CC([O-])=O UEUXEKPTXMALOB-UHFFFAOYSA-J 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention discloses a lysis solution, a DNA extracting solution, a lysis and extraction method, a DNA extraction kit, application and a PCR system. The lysis solution is used for lysing fish tissues to release DNA, and comprises: the lysis solution comprises ethylene diamine tetraacetic acid and/or ethylene diamine tetraacetic acid sodium salt, sodium chloride, sodium dodecyl sulfate, strong base and water, wherein the molar concentration of the ethylene diamine tetraacetic acid and/or the ethylene diamine tetraacetic acid sodium salt is 2-10 mM, the molar concentration of the sodium chloride is 1-10 mM, the mass fraction of the sodium dodecyl sulfate is 0.1-10%, and the molar concentration of hydroxide ions of the strong base is 100-300 mM. Compared with the prior art, the lysis solution can provide a DNA template required by fish tissue PCR amplification in a shorter time, the whole process cannot cause reduction or loss of sample DNA, and the operation is simple and convenient and is easy to master.
Description
Technical field
The present invention relates to gene engineering technology field, in particular to a kind of lysate, Fish Tissue DNA extracting solution, cracking
Method, method, Fish Tissue DNA extraction kit and its application of extracting Fish Tissue DNA, the PCR for expanding Fish Tissue DNA
System.
Background technique
The main method that animal tissue DNA is extracted has phenol-chloroform extraction, isopropanol or the isoamyl alcohol extracting after protease cracking
It takes and SDS method, also the cesium chloride precipitation method, chelex-100 or tween (Tween) method and urea for mitochondrial DNA extraction
Extraction method etc., widely used at present is relatively easy convenient commercial animal tissue DNA extracts kit.Pass through this
The genomic DNA that a little methods obtain, can generally remove the protein and fat in original tissue samples, moreover it is possible to precipitate by alcohols
The reagents such as phenols are removed, gained DNA purity is higher, can satisfy most of requirement of experiment.
Obtain the basis that animal tissue DNA is PCR analysis and other molecular biology researches, these technologies and analysis method
It is applied to many researchs and detection field, such as analysis of genetic diversity, species identification and mesh to core DNA or mitochondrial DNA
Preceding DNA bar code technology analysis etc..But many times according to experiment purpose difference, the requirement to DNA purity is also different, some
More focus on quick, the accurate and sensitivity of DNA extraction process in quick analysis detection.
Fish Tissue has perishable corruption, saves the characteristics of difficulty is greater than other meats.Therefore, Fish Tissue is being extracted
When DNA, it is desirable that extraction process is quick and precisely.However, above-mentioned various method for extracting DNA from animal tissue due to operating process is complicated,
Complex steps, time-consuming, and extracts reagent complexity, tends not to meet the needs of Fish Tissue DNA extraction, sometimes can also be because
Reagent remains and DNA is less than and is difficult to carry out PCR amplification after influencing subsequent PCR amplification process or even micro-example processing, especially
It is difficult to be satisfied with quick detection and identification field.In addition, individual extracting methods also have toxicity such as phenol-chloroform extraction method
By force, the disadvantage big to researcher's Health cost.For a certain specific fish musculature, as fish muscle also there is fat to contain
Measure low, be easily handled, after cell cracking can released dna, scale and fin are similar to the nail of mammal, containing a large amount of
Keratin when carrying out DNA extraction to it, is not required to excessive removal fat, and to remove a large amount of keratin.And existing animal groups
DNA extraction method is knitted to be to remove the protein and fat in tissue.It will be apparent that the extracting method of existing animal tissue DNA
It is unfavorable for fish research.
Therefore, a kind of method that can quick and precisely extract DNA in Fish Tissue is needed, in favor of subsequent quick in fish
Research in detection and identification.
Summary of the invention
The characteristics of for Fish Tissue, the embodiment of the invention discloses a kind of Fish Tissue DNA extracting solution and extracting method,
To achieve the purpose that DNA in rapidly extracting Fish Tissue, to accelerate the research such as quick detection and identification of fish.
The present invention provides a kind of lysate, it is used to crack Fish Tissue and releases DNA, comprising:
Ethylenediamine tetra-acetic acid and/or disodium edta, sodium chloride, lauryl sodium sulfate, highly basic and water,
Wherein for lysate, the molar concentration of ethylenediamine tetra-acetic acid and/or disodium edta is 2~
10mM, the molar concentration of sodium chloride are 1~10mM, and the mass fraction of lauryl sodium sulfate is 0.1~10%, and highly basic
The molar concentration of hydroxide ion is 100~300mM.
Preferably, the molar concentration of the ethylenediamine tetra-acetic acid and/or disodium edta is 3~8mM；The chlorine
The molar concentration for changing sodium is 3~7mM；The mass fraction of the lauryl sodium sulfate is 0.5~5%；The hydrogen-oxygen of the highly basic
The molar concentration of radical ion is 150~250mM.
It is highly preferred that the molar concentration of the ethylenediamine tetra-acetic acid and/or disodium edta is 5mM；The chlorine
The molar concentration for changing sodium is 5mM；The mass fraction of the lauryl sodium sulfate is 1%；The hydroxide ion of the highly basic
Molar concentration is 200mM.
Preferably, the highly basic is selected from NaOH and/or KOH.
The present invention also provides a kind of two-component-type Fish Tissue DNA extracting solutions, including as the above-mentioned of the first component
Lysate and neutralizer as the second component, the neutralizer be include: the pH value containing Tween-20 be 6.4~7.4
Tris-HCl buffer, wherein the mass fraction of the Tween-20 in the neutralizer is 0.02%~0.1%, the neutralizer
In Tris molar concentration be 0.2~1mM.
Preferably, the neutralizer be include: Tris-HCl buffer that the pH value containing Tween-20 is 6.8, wherein institute
The mass fraction for stating the Tween-20 in neutralizer is 0.05%；The molar concentration of Tris in the neutralizer is 0.5mM.
It is highly preferred that the neutralizer also includes at least one of following substance:
Bovine serum albumin(BSA),
Gelatin, and
Dithiothreitol (DTT).
It is highly preferred that the total volume based on the neutralizer, the neutralizer also includes the ox blood of 0.02~0.1mg/mL
At least one of albumin, the gelatin of 0.2~2mg/mL and dithiothreitol (DTT) of 0.02~1mg/mL.
The present invention also provides a kind of cleavage methods, so that Fish Tissue is released DNA using above-mentioned lysate, packet
Include following steps:
Fish Tissue is mixed with lysate, 10~30min is kept in 20~85 DEG C of water-bath, later centrifugal treating,
The supernatant of acquisition is the solution for including DNA.
Preferably, 10~20min is kept in 50~65 DEG C of water-bath.
It is highly preferred that keeping 10min in 55 DEG C of water-bath.
Preferably, the mixed mode is oscillation.
It is highly preferred that the ratio of the Fish Tissue and the lysate is the μ L of 10~25mg:200~400.
It is highly preferred that the ratio of the Fish Tissue and the lysate is 25mg:400 μ L.
The present invention also provides a kind of methods for extracting Fish Tissue DNA, comprising the following steps:
Fish Tissue is mixed with above-mentioned lysate, in 20~85 DEG C of water-bath keep 10~30min, then with it is above-mentioned
Neutralizer mixing, centrifugal treating later, the supernatant of acquisition is the solution containing DNA.
Preferably, 10~20min is kept in 50~65 DEG C of water-bath.
It is highly preferred that keeping 10min in 55 DEG C of water-bath.
It is highly preferred that the volume ratio of the lysate and the neutralizer is 10:3~6.
It is highly preferred that the ratio of the Fish Tissue and the lysate is the μ l of 10~25mg:200~400.
It is highly preferred that the ratio of the Fish Tissue, first component and second component is 25mg:400 μ L:
120μL。
Preferably, the mixed mode is oscillation.
Preferably, the Fish Tissue is selected from: muscle, internal organ, fin, scale or bone.
The present invention also provides a kind of Fish Tissue DNA extraction kits, including above-mentioned lysate and above-mentioned neutralization
Liquid.
It preferably, also include Fish Tissue DNA extraction operation specification.
The present invention also provides above-mentioned kits to extract the application in Fish Tissue DNA.
The present invention also provides a kind of PCR systems for expanding Fish Tissue DNA, which includes by said extracted fish
The obtained solution containing DNA of the method for class loading DNA, the total volume based on the PCR system, the solution containing DNA
Volume fraction be 2%~15%.
It is preferably based on the total volume of the PCR system, the volume fraction of the solution containing DNA is 10%.
The beneficial effect of technical solution provided in an embodiment of the present invention is: compared with prior art, can be in shorter time
DNA profiling needed for interior offer Fish Tissue PCR amplification, whole process not will cause the reduction or loss of sample DNA, and grasp
Make simple and convenient, is easy to grasp.Molecular biology research for fish based on DNA level, such as quick detection and identification research, mentions
More efficiently processing method is supplied.Also, the present invention is using the Various Tissues of fish as the raw material for extracting DNA, certain
The material source of the molecular biology research of fish is expanded in degree.The present invention is based on most basic heating water baths and centrifugation to set
It is standby, it is suitable for most of detection and research unit applications, and time saving and energy saving, is a kind of basis for fast and easily obtaining DNA analysis
Technology.In addition, lysate provided by the invention is almost non-toxic, the health of researcher will not be damaged.
Detailed description of the invention
To describe the technical solutions in the embodiments of the present invention more clearly, make required in being described below to embodiment
Attached drawing is briefly described, it should be apparent that, drawings in the following description are only some embodiments of the invention, for
For those of ordinary skill in the art, without creative efforts, it can also be obtained according to these attached drawings other
Attached drawing.
Fig. 1 is the solution containing DNA that is prepared using A1~A4 of the embodiment of the present invention, B1~B4 and C1~C4 as template solution
The agarose gel electrophoresis figure of the PCR product of acquisition；
Fig. 2 is using C1~C4 of the embodiment of the present invention and the solution containing DNA of phenol-chloroform extraction method preparation as template solution
The agarose gel electrophoresis figure of the PCR product of acquisition.
Specific embodiment
To keep technical solution of the present invention and advantage clearer, below in conjunction with attached drawing to embodiment of the present invention make into
One step it is described in detail.
In the first aspect of the present invention, the embodiment of the invention discloses a kind of lysate, be used to crack Fish Tissue and
Release DNA, comprising: ethylenediamine tetra-acetic acid (EDTA) and/or disodium edta, sodium chloride (NaCl), dodecyl
Sodium sulphate (SDS), highly basic and water, wherein for lysate, ethylenediamine tetra-acetic acid and/or disodium edta
Molar concentration be 2~10mM, the molar concentration of sodium chloride is 1~10mM, the mass fraction of lauryl sodium sulfate is 0.1~
10%, and the hydroxide ion (OH of highly basic-) molar concentration be 100~300mM.
The principle of first aspect present invention are as follows: highly basic and SDS can make cell rupture, protein denaturation, in chromosome
Protein separated with DNA (DNA), to release DNA.In addition to this, the lysate of strong basicity, pH value model
The lysate for enclosing 12~13.5, especially pH value range 12~12.6 can remove a large amount of keratin；Under strongly alkaline conditions,
Lauryl sodium sulfate can be such that protein precipitates.After fish cell is broken, ethylenediamine tetra-acetic acid or ethylenediamine tetra-acetic acid
Sodium salt can the metal ions such as chela and magnesium ion in Fish Tissue or cell and calcium ion, to inhibit deoxyribonuclease pair
The degradation of DNA.Sodium chloride is conducive to improve solubility of the DNA in lysate, increases DNA stability, makes to be easy to DNA's
It saves.
The principle for releasing DNA based on above-mentioned cracking Fish Tissue can be made using lysate provided by the invention
DNA quick release in Fish Tissue comes out, and DNA is not degraded, and obtains quality rapidly and purity is higher contains DNA's
Solution lays the foundation for subsequent molecular biology research.
It should be noted that " fish " described in the present invention are in taxonomy is defined as: fish be body by corneoscute,
It using gill breathing, uses fin as locomotive organ and with the alternating temperature aquatic vertebrate that upper lower jaw is ingested, belongs in Chordata
Vertebrate.
In addition, Fish Tissue described in the present invention can be the groups such as muscle, internal organ, fin, scale and the bone of fish
It knits.Lysate disclosed by the embodiments of the present invention be suitable for any tissue of fish cracking and released dna；Preferably, the flesh of fish
Meat, internal organ, fin, scale and bone；It is further preferred that muscle, internal organ, fin and scale；Still further preferably, muscle
And internal organ, wherein internal organ can be liver；Most preferably, muscle.
Further more, highly basic described in the present invention refers to what alkali soluble ionized completely in water energy generation, belong to highly basic.In the present invention
In, highly basic is preferably inorganic strong alkali, more preferably NaOH and/or KOH.Disodium edta can be ethylenediamine tetra-acetic acid
Disodium.
Next to prepare the molar concentration containing EDTA of 1L as 2mM, the molar concentration of NaCl is 1mM, the quality of SDS
For the molar concentration for the hydroxide ion that score is 0.2%, NaOH is the lysate of 200mM, illustrate lysate of the invention
Preparation method:
Accurately weighed 0.548g EDTA, 0.058g NaCl, 2.017g SDS, 7.999g NaOH are dissolved separately in respectively
In distilled water, it will mixed containing the solution of these four solutes, and be settled to 1L with water.
It will be appreciated by persons skilled in the art that in molecular biology research, water used in reagent preparation is usually
Distilled water, distilled water, deionized water and ultrapure water, wherein being excellent with ultrapure water and deionized water.In the feelings of not specified otherwise
Under condition, used water all refers to distilled water, distilled water deionized water in the embodiment of the present invention.
Lysate in the embodiment of the present invention can be preferred are as follows: ethylenediamine tetra-acetic acid and/or disodium edta
Molar concentration is preferably 3~8mM；The molar concentration of sodium chloride is 3~7mM；The mass fraction of lauryl sodium sulfate be 0.5~
5%；The molar concentration of the hydroxide ion of highly basic is 150~250mM.The lysate of this concentration range can not only obtain high concentration
The DNA of high quality, and remaining reagent has little effect subsequent PCR reaction in the solution containing DNA prepared.
Lysate in the embodiment of the present invention can also be more preferably: ethylenediamine tetra-acetic acid and/or sodium ethylene diamine tetracetate
The molar concentration of salt is 5mM；The molar concentration of sodium chloride is 5mM；The mass fraction of lauryl sodium sulfate is 1%；Highly basic
The molar concentration of hydroxide ion is 200mM.The lysate of this concentration can not only obtain the DNA of higher concentration high quality, and
Remaining reagent has little effect subsequent PCR reaction in the solution containing DNA prepared.
In the second aspect of the present invention, the embodiment of the present invention discloses a kind of cleavage method again, is made using above-mentioned lysate
Fish Tissue cracks and releases DNA, comprising the following steps: mixes Fish Tissue with lysate, in 20~85 DEG C of water-bath
10~30min of middle holding, centrifugal treating, the supernatant of acquisition are the solution containing DNA later.
It is extracted in Fish Tissue from can be seen that in above-mentioned cleavage method using lysate disclosed in first aspect present invention
DNA, operating process is very simple, and the time is short, it can be achieved that rapidly extracting to Fish Tissue DNA.
It should be noted that first having to carry out at sterilizing lysate before extracting Fish Tissue DNA using lysate
Reason.And the method for carrying out sterilization treatment to reagent is conventional technical means in the art, researcher can be according to the item in laboratory
Part voluntarily selects, and therefore, therefore not to repeat here by the present invention.In addition, if researcher be not at once to the supernatant of acquisition into
Supernatant separation can be come out, be saved backup in -20 DEG C by row subsequent experimental.It is also ability for the method for separating supernatant
The customary means of field technique personnel, therefore not to repeat here by the present invention.
In the embodiment of cleavage method, water bath condition is preferred are as follows: 10~20min is kept in 50~65 DEG C of water-bath；
More preferably: keeping 10min in 55 DEG C of water-bath.
In the embodiment of cleavage method, the hybrid mode of Fish Tissue and lysate can be preferred are as follows: oscillation.Oscillation side
Formula can be also possible to artificially vibrate, to realize that Fish Tissue quickly sufficiently connects with lysate to be realized with mini-vibrator
Touching makes Fish Tissue and cell rapid disruption and released dna, to realize DNA rapid extraction.In addition, duration of oscillation can be
Tens seconds, such as 20 seconds, 40 seconds, do not have to more than 60 seconds usually.Certain those skilled in the art can based on practical experience voluntarily
It holds, the present invention is not specifically limited herein.The condition of centrifugation is also the common means of those skilled in the art, can be 10000g
It is centrifuged 2~5min, 5min or more can also be centrifuged with 5000g or more, it is proposed that do not have to more than 10min, the present invention does not make specifically herein
It limits.G described herein is that the power about 9.8N, 10000g that acceleration generates are about 10000 kilograms of centrifugal force.
In the embodiment of cleavage method, it is preferable that the ratio of Fish Tissue and lysate is 10~25mg:200~400
μL；It is highly preferred that the ratio of Fish Tissue and lysate is 25mg:400 μ L.The additional amount of lysate is very few, and supernatant (contains
The solution of DNA) in impurity (predominantly albumen and fat) concentration will increase accordingly, may result in PCR in follow-up study
Reaction is suppressed.Lysate is excessive, can also reduce supernatant (containing DNA while causing reagent unnecessary waste
Solution) in DNA content, to have an adverse effect to follow-up study.
In the third aspect of the present invention, the embodiment of the invention also discloses a kind of two-component-type Fish Tissue DNA extracting solution,
Including the above-mentioned lysate as the first component and as the neutralizer of the second component, neutralizer is to include: containing Tween-20
PH value be 6.4~7.4 Tris-HCl buffer, wherein the mass fraction of the Tween-20 in neutralizer (tween-20) be
The molar concentration of 0.02%~0.1%, the Tris in neutralizer are 0.2~1mM.Wherein, it for the composition of lysate, please refers to
A kind of related content for lysate that first aspect present invention is recorded, therefore not to repeat here by the present invention.
Narration according to a first aspect of the present invention, those skilled in the art it is known that above-mentioned lysate by fish
DNA in class loading is released, and narration according to a second aspect of the present invention, those skilled in the art will also be appreciated that
How solution containing DNA is prepared.During biological study, preparation DNA is an element task, is usually also needed to system
Standby DNA carries out subsequent research work, for example, carry out amplification in vitro to containing a certain specific DNA in DNA solution, i.e., it is logical
Often described polymerase chain reaction (PCR), the various substances in lysate are likely to influence PCR reaction, mainly pass through
The activity of archaeal dna polymerase is reduced to influence PCR reaction.Tris-HCl in neutralizer provides a buffer system for DNA, at this
DNA is in stable state in a system, and Tris-HCl and Tween-20 interaction can be played the role of protecting archaeal dna polymerase, is
PCR reaction provides advantageous guarantee.
In the embodiment of two-component-type Fish Tissue DNA extracting solution, it is preferable that neutralizer is to include: containing Tween-20
PH value be 6.8 Tris-HCl buffer, wherein the mass fraction of the Tween-20 in neutralizer be 0.05%；In neutralizer
Tris molar concentration be 0.5mM.
In the embodiment of two-component-type Fish Tissue DNA extracting solution, it is highly preferred that neutralizer also includes bovine serum albumin
At least one of white (BSA), gelatin and dithiothreitol (DTT) (DTT).Bovine serum albumin(BSA) (BSA), gelatin and dithiothreitol (DTT)
(DTT) any one in or combination, the DNA polymerase activity being more advantageous in protection PCR system, to keep PCR reaction suitable
Benefit carries out.
It should be noted that gelatin is unfixed structure and relative molecular weight, by animal skin, bone, sarolemma, flesh evil spirit
Collagenous portion in equal connective tissues degrades and becomes white or faint yellow, translucent, micro-strip gloss thin slice or powder.This hair
Used gelatin is edible in bright embodiment or the other gelatin of technical grade, and suitable for scientific research impurity content is very
Low high-purity gelatin.
In the embodiment of two-component-type Fish Tissue DNA extracting solution, bovine serum albumin(BSA) (BSA), gelatin and two sulphur Soviet Union
Sugar alcohol (DTT) is further preferred are as follows: the total volume based on neutralizer, neutralizer also include the cow's serum egg of 0.02~0.1mg/mL
At least one of white, 0.2~2mg/mL gelatin and the dithiothreitol (DTT) of 0.02~1mg/mL.
It will be appreciated by persons skilled in the art that protein content should be reduced in the solution containing DNA to the greatest extent, and pass through hair
The research of bright people confirms, a small amount of BSA and/or gelatin are added in the solution of Xiang Hanyou DNA and is advantageous to PCR reaction.
In the fourth aspect of the present invention, above-mentioned two-component-type Fish Tissue is utilized embodiment of the invention discloses a kind of
The method of DNA extracting solution extraction Fish Tissue DNA, comprising the following steps: mix Fish Tissue with above-mentioned lysate, 20
10~30min is kept in~85 DEG C of water-bath, then is mixed with above-mentioned neutralizer, and centrifugal treating, the supernatant of acquisition are later
Solution containing DNA.Wherein, for the composition of lysate and neutralizer, the one kind for please referring to third aspect present invention record is double
Related content in component type Fish Tissue DNA extracting solution, therefore not to repeat here by the present invention.
For the ratio of Fish Tissue and lysate, the hybrid mode of Fish Tissue and lysate, Fish Tissue and cracking
The mixed water bath condition of liquid, those skilled in the art can be with reference in a kind of cleavage methods that second aspect of the present invention is recorded
Related content, therefore not to repeat here by the present invention.
In the embodiment of method for extracting Fish Tissue DNA, it is preferable that the volume ratio of lysate and neutralizer is 10:3
~6.This parameter can not only neutralize the alkalinity of the solution containing DNA, moreover it is possible to give archaeal dna polymerase with strong protection, keep PCR anti-
It should go on smoothly.
In the embodiment of method for extracting Fish Tissue DNA, it is further preferable that Fish Tissue, the first component and second
The ratio of component is 25mg:400 μ L:120 μ L.
It should be further noted that the hybrid mode of Fish Tissue and lysate can be oscillation, neutralizer and water-bath
The hybrid mode of the mixture of lysate and Fish Tissue afterwards can be oscillation.Mode of oscillation and duration of oscillation can be joined
The related content in the embodiment of second aspect of the present invention record is examined, therefore not to repeat here by the present invention.It can be with for centrifugal condition
The related content in embodiment recorded with reference to second aspect of the present invention, therefore not to repeat here by the present invention.
In the fifth aspect of the invention, embodiment of the invention discloses a kind of Fish Tissue DNA extraction kits, including
Above-mentioned lysate and above-mentioned neutralizer.For the composition of lysate and neutralizer, third aspect present invention record is please referred to
A kind of two-component-type Fish Tissue DNA extracting solution in related content, the present invention therefore not to repeat here.
In the embodiment of Fish Tissue DNA extraction kit of the invention, a kind of Fish Tissue DNA extraction kit is also
Include Fish Tissue DNA extraction operation specification.For the operating procedure recorded in Fish Tissue DNA extraction operation specification,
It please refers to a kind of of fourth aspect present invention record and extracts Fish Tissue using above-mentioned two-component-type Fish Tissue DNA extracting solution
The method of DNA, therefore not to repeat here by the present invention.
For example, Fish Tissue DNA extraction operation specification can be with are as follows: is washed with deionized water Fish Tissue, takes
25mg Fish Tissue after shredding, is put into centrifuge tube, and the lysate of 400 μ L is added, and is vibrated 20 seconds, later in 55 DEG C of water-bath
Middle holding 10min adds 120 μ L neutralizers, vibrates 20 seconds, and last 10000g is centrifuged 2min, takes supernatant, as contains
The solution of DNA.
In the sixth aspect of the present invention, embodiment of the invention discloses above-mentioned Fish Tissue DNA extraction kits to mention
Take the application in Fish Tissue DNA.
A kind of Fish Tissue DNA extraction kit that sixth aspect present invention is recorded can be based on fish DNA molecular water
Any research field application on flat.For example, the quick detection of Fish genomes, identification of fish species etc..The research of fish
Personnel can need voluntarily to select according to research, and the present invention is numerous to list herein.
In the seventh aspect of the present invention, embodiment of the invention discloses a kind of PCR system for expanding Fish Tissue DNA,
The PCR system includes the obtained solution containing DNA of method by said extracted Fish Tissue DNA, based on the PCR system
Total volume, the volume fraction of the solution containing DNA are 2%~15%.The PCR system can be with are as follows:
In the embodiment of the PCR system of amplification Fish Tissue DNA, it is preferable that the volume fraction of the solution containing DNA is
10%.
It should be noted that described in the present invention it is accurately weighed mean weigh weight should be accurately to thousand points of weighed weight
One of.MM is expressed as mmol/L.
Reagent, material and instrument
NaOH, Tris-HCL, EDTA, NaCL, SDS and Tween-20 are domestic analytical reagents；
Taq archaeal dna polymerase is originated from Thermo Fischer Scient Inc. (Thermo Fisher Scientific)；
DNAMarker is Shanghai Sheng Gong Bioisystech Co., Ltd product.
Test material is muscle, liver, the middle body vertebrae, squama of little yellow croaker (Larimichthys polyactis)
Piece and fin.
18S rRNA gene primer sequence is derived from bibliography: Fajardo V, Gonzalez I, Martin I, et
al.Real-time PCR for detection and quantification of red deer(Cervus
elaphus),fallow deer(Dama dama),and roe deer(Capreolus capreolus)in meat
mixtures.Meat Sci,2008,79(2):289-298.
I gene primer sequence of CO is derived from bibliography: Ward, R.D., T.S.Zemlak, B.H.Innes,
P.R.Last,P.D.N.Hebert.DNA barcoding Australia's fish species.Philosophical
Transactions of the Royal Society B:Biological Sciences,2005,360:1847-1857.
18S rRNA gene and I gene primer sequence of primer sequence and CO are all by Hangzhou Qing Ke Bioisystech Co., Ltd
Synthesis.
Ultraviolet light analyzer Nanodrop 2000, Thermo Fischer Scient Inc.；
PCR instrument MJ PTC200, gene instrument company, the U.S.；
Gel imaging system, Liuyi Instruments Plant, Beijing.
Embodiment A1~A4, embodiment B1~B4 and embodiment C1~C4
The extraction of DNA in little yellow croaker tissue.Wherein used little yellow croaker tissue is respectively scale, bone, muscle, liver
It is dirty.
Embodiment A1~A4
Little yellow croaker tissue is cleaned with the distilled water of sterilizing, the little yellow croaker tissue after taking 25mg to clean is put into 1.5mL after shredding
In centrifuge tube, the lysate of 400 μ L is added, vibrates 20 seconds, keeps 10min in 55 DEG C of water-bath, adds 120 μ L neutralization
Liquid vibrates 20 seconds, and then 10000g is centrifuged 2min, takes supernatant to get the solution containing DNA.
The difference of the reaction condition of embodiment A1~A4 and embodiment B1~B4 and embodiment C1~C4 is only that lysate
It is different with the respective reagent proportion in neutralizer, it is shown in Table 1:
The composition of reagent in 1 lysate of table and neutralizer
The different tissues of digital representation little yellow croaker in embodiment A1~A4, embodiment B1~B4 and embodiment C1~C4,
Wherein 1, scale, 2, bone, 3, muscle, 4, liver.
It is dense that DNA is carried out to the solution containing DNA of embodiment A1~A4, embodiment B1~B4 and embodiment C1~C4 preparation
Degree measurement, the results are shown in Table 2:
2 DNA concentration of table
From Table 2, it can be seen that for same tissue, used extraction in embodiment B1~B4 and embodiment C1~C4
DNA concentration is without significant difference in the solution containing DNA of liquid preparation, and the preparation of used extracting solution contains in embodiment A1~A4
There is DNA concentration in the solution of DNA to be significantly lower than the former two.
Using the solution for containing DNA prepared by embodiment A1~A4, embodiment B1~B4 and embodiment C1~C4 as DNA mould
Plate solution expands Eukaryotic 18S rRNA gene, it is contemplated that amplified production 140bp, 18S rRNA gene primer sequence is such as
Under:
Upstream primer: 5'-TCTGCCCTATCAACTTTCGATGG-3'
Downstream primer: 5'-TAATTTGCGCGCCTGCTG-3'
PCR system total volume is 20 μ L, as shown in table 3 below containing reagent:
Each reagent dosage and final concentration in 3 18S rRNA gene PCR system of table
PCR response procedures are as follows: 95 DEG C of denaturation 5min；95 DEG C of denaturation 30s, 59 DEG C of annealing 30s, 72 DEG C of extension 30s are one
Circulation amounts to 30 circulations；Then 72 DEG C of holding 10min.It is cooled to 4 DEG C after reaction.
Each 6 μ L of PCR product is detected through 2% agarose gel electrophoresis, nucleic acid dye Gel Red dyeing, gel imaging system
Analysis, as a result as shown in Figure 1 (M in Fig. 1 indicates DNAMarker).As shown in Figure 1, primer size is consistent with expected 140bp,
Illustrate that amplification has obtained 18S rRNA gene product.Electrophoresis result can be used as the basic foundation of molecular biological analysis.From small
The DNA that the different tissues of yellow croaker extract carries out PCR, and from the point of view of electrophoresis result, the effect of bone, scale and fin is than muscle and liver
It is slightly worse.DNA extraction effect in muscle and liver organization is preferable, the two no significant difference.That is, fish provided by the invention
Class loading DNA extracting solution and extracting method can be realized the DNA extraction to fish different tissues, imitate to the extraction of muscle and liver
Fruit is better than the extraction effect to bone, scale and fin.
In conjunction with from the point of view of the result of table 2 and Fig. 1, for the same tissue of little yellow croaker, embodiment B1~B4 and embodiment C1~
DNA concentration is and identical in PCR reaction condition without significant difference in the solution containing DNA of used extracting solution preparation in C4
In the case where, the electrophoresis result of PCR product, which is but shown, to be carried out using the DNA solution of embodiment B1~B4 preparation as DNA profiling solution
The PCR product amount that PCR is obtained is considerably less than embodiment C1~C4, traces it to its cause, it may be possible to used in embodiment B1~B4
Each reagent content is high in extracting solution, it is suppressed that PCR reaction.
Embodiment D~embodiment S
Embodiment D~embodiment S is identical as Fish Tissue DNA extracting solution used in embodiment C1~C4, and difference is
Little yellow croaker tissue types, the dosage and water bath condition of little yellow croaker tissue, lysate and neutralizer are different.
| Embodiment | Tissue types | It organizes (mg) | Lysate (μ L) | Neutralizer (μ L) | Bath temperature (DEG C) | Water bath time (min) |
| D | Muscle | 25 | 400 | 120 | 55 | 10 |
| E | Muscle | 10 | 200 | 60 | 50 | 20 |
| F | Muscle | 25 | 200 | 100 | 75 | 30 |
| H | Liver | 25 | 400 | 120 | 55 | 10 |
| I | Liver | 10 | 200 | 60 | 50 | 20 |
| G | Liver | 25 | 200 | 100 | 75 | 30 |
| K | Bone | 25 | 400 | 120 | 55 | 10 |
| L | Bone | 10 | 200 | 60 | 50 | 20 |
| M | Bone | 25 | 200 | 100 | 75 | 30 |
| N | Fin | 25 | 400 | 120 | 55 | 10 |
| O | Fin | 10 | 200 | 60 | 50 | 20 |
| P | Fin | 25 | 200 | 100 | 75 | 30 |
| Q | Scale | 25 | 400 | 120 | 55 | 10 |
| R | Scale | 10 | 200 | 60 | 50 | 20 |
| S | Scale | 25 | 200 | 100 | 75 | 30 |
DNA concentration measurement is carried out to solution of the embodiment D~embodiment S preparation containing DNA, inventors have found that processing
The DNA concentration in the solution containing DNA that muscle and liver obtain is 50~200ng/ μ L, and what processing fin obtained contains DNA
Solution in DNA concentration be 20~100ng/ μ L, processing scale obtain the solution containing DNA in DNA concentration 8
~40ng/ μ L.And A in the above-mentioned solution containing DNA260/A280Ratio between 1.2~1.6, illustrate reality of the invention
The DNA purity for applying example acquisition is good.
Comparative example
The DNA in little yellow croaker muscle is extracted using phenol-chloroform extraction method.Wherein, phenol-chloroform extraction method is referring to " molecule gram
Grand experiment guide " third edition, the first volume: 463-485.
By DNA and embodiment C1~C4 are obtained in the little yellow croaker muscle extracted using phenol-chloroform extraction method DNA simultaneously into
Row PCR experiment, to verify the extracting method of Fish Tissue DNA of the invention.
Expand the Mitochondrial cytochrome c oxidase I (cytochrome coxidase I, CO I) of little yellow croaker DNA bar code
Gene, length 658bp, primer sequence are as follows:
I upstream region of gene primer of CO: 5'-TCAACCAACCACAAAGACATTGGCAC-3'
I downstream of gene primer of CO: 5'-TAGACTTCTGGGTGGCCAAAGAATCA-3'
PCR system total volume is 20 μ L, and it is as follows that PCR system contains reagent: the Buffer buffer of 2.0 μ L, upstream and downstream
Each 2 μ L of primer, the dNTP mixture of 1.6 μ L, the MgCl of 1.6 μ L2Buffer, 2.0 μ L DNA profiling solution, 0.2 μ L Taq
Archaeal dna polymerase, finally plus water is supplemented to 20 μ L.
I gene PCR response procedures of CO are as follows: 95 DEG C of denaturation 5min；95 DEG C of denaturation 30sec, 55 DEG C of annealing 35sec, 72 DEG C are prolonged
Stretching 35sec is a circulation, amounts to 30 circulations；Then 72 DEG C of holding 10min.After be cooled to 4 DEG C.
Each 6 μ L of PCR product is detected through 2% agarose gel electrophoresis, and nucleic acid dye Gel Red dyeing, gel systems are taken pictures,
As a result (1,2,3 and 4 in Fig. 2 respectively indicate using the muscle of little yellow croaker, liver, scale and bone as sample acquisition as shown in Figure 2
PCR product, 5 indicate using phenol-chloroform extraction method processing little yellow croaker muscle samples obtain PCR product, M indicate
DNAMarker).As shown in Figure 2, the muscle, liver, scale and bone samples of processing obtain the special of expected size 658bp
Property band, muscle, liver, the DNA extraction effect in scale and the DNA that is extracted using phenol-chloroform are essentially identical, the effect of bone
It is slightly worse.The quality and content for illustrating the DNA obtained using the method for extraction Fish Tissue DNA of the invention are all no less than phenol-
The DNA that chloroform extraction obtains.Moreover, relative to phenol-chloroform extraction method, Fish Tissue DNA extraction method of the invention
Easy to operate, time-consuming short, the toxicity of extracting solution is low.In addition, utilizing Fish Tissue DNA extracting solution of the invention and extracting method system
The gene that standby DNA profiling solution out directly expands long segment also has good effect.
It should be noted that Canada scientist Hebert proposes CO in proposition DNA bar code technology in 2003, in the same year
I gene is as bar code target.DNA bar code technology selects mark of one section of species specificity sequence as species identification, CO I
Gene is used as target application so far from proposition, important function has been played in animals and plants and microbial identification and species identification, especially
It shows unrivaled superiority in the identification of fish, is that fish identify most potential tool, and go back in the world
There is the database of special fish DNA bar code, provides important support for the Rapid identification of fish.
Technical solution 1
A kind of lysate is used to crack Fish Tissue and releases DNA characterized by comprising
Ethylenediamine tetra-acetic acid and/or disodium edta, sodium chloride, lauryl sodium sulfate, highly basic and water,
Wherein for lysate, the molar concentration of ethylenediamine tetra-acetic acid and/or disodium edta is 2~
10mM, the molar concentration of sodium chloride are 1~10mM, and the mass fraction of lauryl sodium sulfate is 0.1~10%, and highly basic
The molar concentration of hydroxide ion is 100~300mM.
Technical solution 2
Lysate according to technical solution 1, which is characterized in that
The molar concentration of the ethylenediamine tetra-acetic acid and/or disodium edta is 3~8mM；The sodium chloride
Molar concentration is 3~7mM；The mass fraction of the lauryl sodium sulfate is 0.5~5%；The hydroxide ion of the highly basic
Molar concentration be 150~250mM.
Technical solution 3
Lysate according to technical solution 1, which is characterized in that
The molar concentration of the ethylenediamine tetra-acetic acid and/or disodium edta is 5mM；Mole of the sodium chloride
Concentration is 5mM；The mass fraction of the lauryl sodium sulfate is 1%；The molar concentration of the hydroxide ion of the highly basic is
200mM。
Technical solution 4
According to the described in any item lysates of technical solution 1 to 3, which is characterized in that the highly basic be selected from NaOH and/or
KOH。
Technical solution 5
A kind of two-component-type Fish Tissue DNA extracting solution, which is characterized in that including the technical solution 1 as the first component
~4 described in any item lysates and neutralizer as the second component, the neutralizer be include: the pH containing Tween-20
Value be 6.4~7.4 Tris-HCl buffer, wherein the mass fraction of the Tween-20 in the neutralizer be 0.02%~
The molar concentration of 0.1%, the Tris in the neutralizer are 0.2~1mM.
Technical solution 6
According to DNA extracting solution described in technical solution 5, which is characterized in that the neutralizer is to include: containing Tween-20
PH value be 6.8 Tris-HCl buffer, wherein the mass fraction of the Tween-20 in the neutralizer be 0.05%；It is described
The molar concentration of Tris in neutralizer is 0.5mM.
Technical solution 7
The DNA extracting solution according to technical solution 5 or 6, which is characterized in that the neutralizer also includes in following substance
At least one:
Bovine serum albumin(BSA),
Gelatin, and
Dithiothreitol (DTT).
Technical solution 8
According to DNA extracting solution described in technical solution 7, which is characterized in that the total volume based on the neutralizer, it is described in
It also include the two sulphur Soviet Union of the bovine serum albumin of 0.02~0.1mg/mL, the gelatin of 0.2~2mg/mL and 0.02~1mg/mL with liquid
At least one of sugar alcohol.
Technical solution 9
A kind of cleavage method releases Fish Tissue using such as the described in any item lysates of technical solution 1 to 4
DNA, which comprises the following steps:
Fish Tissue is mixed with lysate, 10~30min is kept in 20~85 DEG C of water-bath, later centrifugal treating,
The supernatant of acquisition is the solution for including DNA.
Technical solution 10
According to method described in technical solution 9, which is characterized in that keep 10~20min in 50~65 DEG C of water-bath.
Technical solution 11
According to method described in technical solution 10, which is characterized in that keep 10min in 55 DEG C of water-bath.
Technical solution 12
According to method described in technical solution 9, which is characterized in that the mixed mode is oscillation.
Technical solution 13
According to method described in technical solution 9, which is characterized in that the ratio of the Fish Tissue and the lysate is 10
The μ of~25mg:200~400 L.
Technical solution 14
According to method described in technical solution 13, which is characterized in that the ratio of the Fish Tissue and the lysate is
25mg:400μL。
Technical solution 15
A method of extracting Fish Tissue DNA, which comprises the following steps:
Fish Tissue is mixed with the described in any item lysates of technical solution 5 to 8, is kept in 20~85 DEG C of water-bath
10~30min, then mixed with the described in any item neutralizers of technical solution 5 to 8, centrifugal treating, the supernatant of acquisition are later
For the solution containing DNA.
Technical solution 16
According to method described in technical solution 15, which is characterized in that keep 10~20min in 50~65 DEG C of water-bath.
Technical solution 17
According to method described in technical solution 16, which is characterized in that keep 10min in 55 DEG C of water-bath.
Technical solution 18
According to the described in any item methods of technical solution 15 to 17, which is characterized in that the lysate and the neutralizer
Volume ratio be 10:3~6.
Technical solution 19
According to method described in technical solution 18, the ratio of the Fish Tissue and the lysate is 10~25mg:200
~400 μ l.
Technical solution 20
According to method described in technical solution 19, which is characterized in that the Fish Tissue, first component and described
The ratio of two components is 25mg:400 μ L:120 μ L.
Technical solution 21
According to method described in technical solution 15, which is characterized in that the mixed mode is oscillation.
Technical solution 22
According to method described in technical solution 15, which is characterized in that the Fish Tissue is selected from: muscle, internal organ, fin,
Scale or bone.
Technical solution 23
A kind of Fish Tissue DNA extraction kit, which is characterized in that including the described in any item cracking of technical solution 1 to 4
Liquid and the described in any item neutralizers of technical solution 5 to 8.
Technical solution 24
According to Fish Tissue DNA extraction kit described in technical solution 23, which is characterized in that also include Fish Tissue
DNA extraction operation specification.
Technical solution 25
Kit described in technical solution 23 or 24 is extracting the application in Fish Tissue DNA.
Technical solution 26
A kind of PCR system expanding Fish Tissue DNA, which is characterized in that the PCR system includes by technical solution 15 to 22
The obtained solution containing DNA of any one the method, the total volume based on the PCR system, the solution containing DNA
Volume fraction is 2%~15%.
Technical solution 27
According to PCR system described in technical solution 26, which is characterized in that the total volume based on the PCR system, it is described to contain
The volume fraction of the solution of DNA is 10%.
The above is merely for convenience of it will be understood by those skilled in the art that technical solution of the present invention, not to limit
The present invention.All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should be included in this
Within the protection scope of invention.
Claims (18)
1. a kind of two-component-type Fish Tissue DNA extracting solution, which is characterized in that including as the first component lysate and conduct
The neutralizer of second component,
The lysate is composed of the following components:
Ethylenediamine tetra-acetic acid and/or disodium edta, sodium chloride, lauryl sodium sulfate, highly basic and water,
Wherein for lysate, the molar concentration of ethylenediamine tetra-acetic acid and/or disodium edta is 2~
10mM, the molar concentration of sodium chloride are 1~10mM, and the mass fraction of lauryl sodium sulfate is 0.1~10%, and highly basic
The molar concentration of hydroxide ion is 100~300mM；
The neutralizer includes: the Tris-HCl buffer that the pH value containing Tween-20 is 6.4~7.4, wherein the neutralizer
In the mass fraction of Tween-20 be 0.02%~0.1%, the molar concentration of the Tris in the neutralizer is 0.2~1mM.
2. two-component-type Fish Tissue DNA extracting solution according to claim 1, which is characterized in that
The molar concentration of the ethylenediamine tetra-acetic acid and/or disodium edta is 3~8mM；Mole of the sodium chloride
Concentration is 3~7mM；The mass fraction of the lauryl sodium sulfate is 0.5~5%；The hydroxide ion of the highly basic rubs
Your concentration is 150~250mM.
3. two-component-type Fish Tissue DNA extracting solution according to claim 1, which is characterized in that
The molar concentration of the ethylenediamine tetra-acetic acid and/or disodium edta is 5mM；The molar concentration of the sodium chloride
For 5mM；The mass fraction of the lauryl sodium sulfate is 1%；The molar concentration of the hydroxide ion of the highly basic is
200mM。
4. two-component-type Fish Tissue DNA extracting solution according to any one of claims 1 to 3, which is characterized in that
The highly basic is selected from NaOH and/or KOH.
5. DNA extracting solution according to claim 4, which is characterized in that the neutralizer includes: the pH containing Tween-20
The Tris-HCl buffer that value is 6.8, wherein the mass fraction of the Tween-20 in the neutralizer is 0.05%；The neutralization
The molar concentration of Tris in liquid is 0.5mM.
6. DNA extracting solution according to claim 4, which is characterized in that the neutralizer also include in following substance extremely
Few one kind:
Bovine serum albumin(BSA),
Gelatin, and
Dithiothreitol (DTT).
7. DNA extracting solution according to claim 6, which is characterized in that the total volume based on the neutralizer, the neutralization
Liquid also includes the two sulphur threoses of the bovine serum albumin of 0.02~0.1mg/mL, the gelatin of 0.2~2mg/mL and 0.02~1mg/mL
At least one of alcohol.
8. a kind of method for extracting Fish Tissue DNA, which comprises the following steps:
Fish Tissue is mixed with the described in any item lysates of claim 1 to 7, in 20~85 DEG C of water-bath keep 10~
30min, then mixed with the described in any item neutralizers of claim 1 to 7, centrifugal treating, the supernatant of acquisition are to contain later
There is the solution of DNA.
9. according to the method described in claim 8, it is characterized in that, keeping 10~20min in 50~65 DEG C of water-bath.
10. according to the method described in claim 9, it is characterized in that, keeping 10min in 55 DEG C of water-bath.
11. according to the described in any item methods of claim 8 to 10, which is characterized in that the lysate and the neutralizer
Volume ratio is 10:3~6.
12. according to the method for claim 11, the ratio of the Fish Tissue and the lysate is 10~25mg:200
~400 μ l.
13. according to the method for claim 12, which is characterized in that the Fish Tissue, first component and described the
The ratio of two components is 25mg:400 μ L:120 μ L.
14. according to the method described in claim 8, it is characterized in that, the mixed mode is oscillation.
15. according to the method described in claim 8, it is characterized in that, the Fish Tissue is selected from: muscle, internal organ, fin, squama
Piece or bone.
16. a kind of Fish Tissue DNA extraction kit, which is characterized in that including the described in any item cracking of claim 1 to 7
Liquid and the described in any item neutralizers of claim 1 to 7.
17. Fish Tissue DNA extraction kit according to claim 16, which is characterized in that also include Fish Tissue DNA
Extraction operation specification.
18. kit described in claim 16 or 17 is extracting the application in Fish Tissue DNA.
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| CN107475455A (en) * | 2017-09-25 | 2017-12-15 | 北京大有泰莱生物技术有限公司 | NDV real-time fluorescence quantitative PCR detection method and its kit |
| CN108795924A (en) * | 2017-12-06 | 2018-11-13 | 宁夏农林科学院 | A kind of quick, simple plant genome DNA extracting method |
| CN109762810A (en) * | 2019-03-22 | 2019-05-17 | 中国水产科学研究院淡水渔业研究中心 | Fish scale DNA rapid extracting method for the analysis of high-throughput microsatellite |
| CN112725333A (en) * | 2021-02-07 | 2021-04-30 | 苏州大学 | Method for rapidly extracting animal genome DNA |
| CN114276932A (en) * | 2022-02-12 | 2022-04-05 | 合肥巅峰生物科技有限公司 | Microbial cell lysate |
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| US6037464A (en) * | 1995-09-27 | 2000-03-14 | Kim; Dong-Soo | Method for the extraction of genomic DNA from fish blood or sperm |
| JP2007124926A (en) * | 2005-11-02 | 2007-05-24 | Visionbio Corp | Dna extractant and method for extracting dna using the same |
| CN103430936A (en) * | 2013-09-06 | 2013-12-11 | 中国水产科学研究院黑龙江水产研究所 | Preparation method of fish fin tissues for extracting genomic DNA (deoxyribonucleic acid) of fish |
| CN103911366A (en) * | 2014-03-19 | 2014-07-09 | 华南理工大学 | Genome DNA extraction method and its kit |
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|---|---|---|---|---|
| US6037464A (en) * | 1995-09-27 | 2000-03-14 | Kim; Dong-Soo | Method for the extraction of genomic DNA from fish blood or sperm |
| JP2007124926A (en) * | 2005-11-02 | 2007-05-24 | Visionbio Corp | Dna extractant and method for extracting dna using the same |
| CN103430936A (en) * | 2013-09-06 | 2013-12-11 | 中国水产科学研究院黑龙江水产研究所 | Preparation method of fish fin tissues for extracting genomic DNA (deoxyribonucleic acid) of fish |
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