CN106282160A - Lysate, extracting solution, cracking and extracting method, test kit and application, PCR system - Google Patents
Lysate, extracting solution, cracking and extracting method, test kit and application, PCR system Download PDFInfo
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Abstract
The invention discloses a kind of lysate, DNA extraction liquid, cracking and extracting method, DNA extraction kit and application, PCR system.This lysate, DNA is discharged for cracking Fish Tissue, including: ethylenediaminetetraacetic acid and/or disodium edta, sodium chloride, sodium lauryl sulphate, highly basic and water, wherein for lysate, the molar concentration of ethylenediaminetetraacetic acid and/or disodium edta is 2～10mM, the molar concentration of sodium chloride is 1～10mM, and the mass fraction of sodium lauryl sulphate is 0.1～10%, and the molar concentration of the hydroxide ion of highly basic is 100～300mM.Compared with prior art, the lysate of the present invention can provide the DNA profiling needed for Fish Tissue PCR amplification in shorter time, and whole process does not results in minimizing or the loss of sample DNA, and simple to operation, it is easy to grasps.
Description
Technical field
The present invention relates to gene engineering technology field, particularly to a kind of lysate, Fish Tissue DNA extraction liquid, cracking
Method, the extraction method of Fish Tissue DNA, Fish Tissue DNA extraction kit and application, the PCR of amplification Fish Tissue DNA
System.
Background technology
The main method of animal tissue's DNA extraction has the phenol-chloroform after protease cracking to extract, isopropanol or isoamyl alcohol extraction
Take and SDS method, also have the cesium chloride sedimentation method, chelex-100 or tween (Tween) method and carbamide extracted for mitochondrial DNA
Extraction methods etc., the most widely used is that relatively easy convenient commercial animal tissue DNA extracts test kit.By this
The genomic DNA that a little methods obtain, typically can remove the protein in original tissue samples and fat, moreover it is possible to precipitated by alcohols
Removing the reagent such as phenols, gained DNA purity is higher, it is possible to meet major part requirement of experiment.
Obtain the basis that animal tissue DNA is pcr analysis and other molecular biology researches, these technology and the method for analysis
It is applied to many research and detection field, such as analysis of genetic diversity, species identification and mesh to core DNA or mitochondrial DNA
Front DNA bar code technical Analysis etc..But many times different according to experiment purpose, the requirement to DNA purity is the most different, at some
Quickly analyze in detection and more focus on the quick, accurate of DNA extraction process and susceptiveness.
Fish Tissue has perishable corruption, preserves the difficulty feature more than other meats.Therefore, Fish Tissue is being extracted
During DNA, it is desirable to extract process quick and precisely.But, above-mentioned various method for extracting DNA from animal tissues owing to operating process is complicated,
Complex steps, the longest, and it is complicated to extract reagent, tend not to meet the needs of Fish Tissue DNA extraction, the most also can be because of
Reagent remains and affects subsequent PCR amplification process, and after even micro-example processes, DNA insufficient total amount is difficult to PCR amplification, especially
It is difficult to be satisfied with quickly detection and qualification field.Additionally, indivedual extracting method, such as phenol-chloroform extraction method, also there is toxicity
By force, big to researcher Health cost shortcoming.For a certain specific fish musculature, also there is such as fish muscle fat and contain
Measure low, it is easy to process, get final product released dna, scale and fin after cell cracking and be similar to the fingernail of mammal, containing substantial amounts of
Keratin, when it is carried out DNA extraction, is not required to too much removal fat, and to remove substantial amounts of keratin.And existing animal groups
Knit DNA extraction method to be to remove the protein in tissue and fat.It will be apparent that the extracting method of existing animal tissue DNA
It is unfavorable for that Fish are studied.
Therefore, need badly and a kind of can quick and precisely extract the method for DNA in Fish Tissue, be beneficial to follow-up quick Fish
Research in detection and qualification.
Summary of the invention
For the feature of Fish Tissue, the embodiment of the invention discloses a kind of Fish Tissue DNA extraction liquid and extracting method,
To reach the purpose of DNA in rapid extraction Fish Tissue, thus accelerate Fish and quickly detect and the research such as qualification.
The invention provides a kind of lysate, it is used for cracking Fish Tissue and discharges DNA, including:
Ethylenediaminetetraacetic acid and/or disodium edta, sodium chloride, sodium lauryl sulphate, highly basic and water,
Wherein for lysate, the molar concentration of ethylenediaminetetraacetic acid and/or disodium edta be 2～
10mM, the molar concentration of sodium chloride is 1～10mM, and the mass fraction of sodium lauryl sulphate is 0.1～10%, and highly basic
The molar concentration of hydroxide ion is 100～300mM.
Preferably, the molar concentration of described ethylenediaminetetraacetic acid and/or disodium edta is 3～8mM；Described chlorine
The molar concentration changing sodium is 3～7mM；The mass fraction of described sodium lauryl sulphate is 0.5～5%；The hydrogen-oxygen of described highly basic
The molar concentration of radical ion is 150～250mM.
It is highly preferred that the molar concentration of described ethylenediaminetetraacetic acid and/or disodium edta is 5mM；Described chlorine
The molar concentration changing sodium is 5mM；The mass fraction of described sodium lauryl sulphate is 1%；The hydroxide ion of described highly basic
Molar concentration is 200mM.
Preferably, described highly basic is selected from NaOH and/or KOH.
Present invention also offers a kind of two-component-type Fish Tissue DNA extraction liquid, above-mentioned including as the first component
Lysate and the neutralizer as second component, described neutralizer is to include: the pH value containing tween 20 is 6.4～7.4
Tris-HCl buffer, the mass fraction of the tween 20 in wherein said neutralizer is 0.02%～0.1%, described neutralizer
In the molar concentration of Tris be 0.2～1mM.
Preferably, described neutralizer is to include: the pH value containing tween 20 is the Tris-HCl buffer of 6.8, Qi Zhongsuo
The mass fraction stating the tween 20 in neutralizer is 0.05%；The molar concentration of the Tris in described neutralizer is 0.5mM.
It is highly preferred that described neutralizer also comprises at least one in following material:
Bovine serum albumin,
Gelatin, and
Dithiothreitol, DTT.
It is highly preferred that cumulative volume based on described neutralizer, described neutralizer also comprises the Sanguis Bovis seu Bubali of 0.02～0.1mg/mL
The gelatin of albumin, 0.2～2mg/mL and 0.02～1mg/mL dithiothreitol, DTT at least one.
Present invention also offers a kind of cleavage method, it utilizes above-mentioned lysate to make Fish Tissue discharge DNA, bag
Include following steps:
Fish Tissue is mixed with lysate, holding 10～30min in the water-bath of 20～85 DEG C, centrifugal treating afterwards,
The supernatant obtained is the solution comprising DNA.
Preferably, in the water-bath of 50～65 DEG C, keep 10～20min.
It is highly preferred that keep 10min in the water-bath of 55 DEG C.
Preferably, the mode of described mixing is vibration.
It is highly preferred that the ratio of described Fish Tissue and described lysate is 10～25mg:200～400 μ L.
It is highly preferred that the ratio of described Fish Tissue and described lysate is 25mg:400 μ L.
Present invention also offers a kind of method extracting Fish Tissue DNA, comprise the following steps:
Fish Tissue is mixed with above-mentioned lysate, the water-bath of 20～85 DEG C keeps 10～30min, then with above-mentioned
Neutralizer mixing, centrifugal treating afterwards, it is thus achieved that supernatant be the solution containing DNA.
Preferably, in the water-bath of 50～65 DEG C, keep 10～20min.
It is highly preferred that keep 10min in the water-bath of 55 DEG C.
It is highly preferred that the volume ratio of described lysate and described neutralizer is 10:3～6.
It is highly preferred that the ratio of described Fish Tissue and described lysate is 10～25mg:200～400 μ l.
It is highly preferred that the ratio of described Fish Tissue, described first component and described second component is 25mg:400 μ L:
120μL。
Preferably, the mode of described mixing is vibration.
Preferably, described Fish Tissue is selected from: muscle, internal organs, fin, scale or skeleton.
Present invention also offers a kind of Fish Tissue DNA extraction kit, including above-mentioned lysate and above-mentioned neutralization
Liquid.
Preferably, Fish Tissue DNA extraction operating instruction is also comprised.
Present invention also offers the application in extracting Fish Tissue DNA of the above-mentioned test kit.
Present invention also offers a kind of PCR system expanding Fish Tissue DNA, this PCR system includes by said extracted fish
The solution containing DNA obtained by the method for class loading DNA, cumulative volume based on this PCR system, the described solution containing DNA
Volume fraction be 2%～15%.
Being preferably based on the cumulative volume of this PCR system, the volume fraction of the described solution containing DNA is 10%.
The technical scheme that the embodiment of the present invention provides provides the benefit that: compared with prior art, can be at shorter time
DNA profiling needed for interior offer Fish Tissue PCR amplification, whole process does not results in minimizing or the loss of sample DNA, and grasps
Make simple and convenient, it is easy to grasp.For Fish molecular biology research based on DNA level, as quickly detected and identification research, carry
Supply processing method the most efficiently.Further, the present invention utilizes the Various Tissues raw material as extraction DNA of Fish, necessarily
The material source of the molecular biology research of Fish is expanded in degree.The present invention sets with centrifugal based on most basic heating in water bath
Standby, be suitable to great majority detection and research unit applies, and time saving and energy saving, it is a kind of basis obtaining DNA analysis fast and easily
Technology.Additionally, the lysate that the present invention provides is almost non-toxic, the health of researcher will not be damaged.
Accompanying drawing explanation
For the technical scheme being illustrated more clearly that in the embodiment of the present invention, in embodiment being described below required for make
Accompanying drawing be briefly described, it should be apparent that, below describe in accompanying drawing be only some embodiments of the present invention, for
From the point of view of those of ordinary skill in the art, on the premise of not paying creative work, it is also possible to obtain other according to these accompanying drawings
Accompanying drawing.
Fig. 1 is that the solution containing DNA prepared with embodiment of the present invention A1～A4, B1～B4 and C1～C4 is as template solution
The agarose gel electrophoresis figure of the PCR primer obtained；
Fig. 2 is that the solution containing DNA prepared with embodiment of the present invention C1～C4 and phenol-chloroform extraction method is as template solution
The agarose gel electrophoresis figure of the PCR primer obtained.
Detailed description of the invention
For making technical scheme and advantage clearer, below in conjunction with accompanying drawing embodiment of the present invention made into
One step ground describes in detail.
In a first aspect of the present invention, the embodiment of the invention discloses a kind of lysate, its be used for cracking Fish Tissue and
Discharge DNA, including: ethylenediaminetetraacetic acid (EDTA) and/or disodium edta, sodium chloride (NaCl), dodecyl
Sodium sulfate (SDS), highly basic and water, wherein for lysate, ethylenediaminetetraacetic acid and/or disodium edta
Molar concentration be 2～10mM, the molar concentration of sodium chloride is 1～10mM, the mass fraction of sodium lauryl sulphate be 0.1～
10%, and the hydroxide ion (OH of highly basic-) molar concentration be 100～300mM.
The principle of first aspect present invention is: highly basic and SDS can make cell rupture, and protein denaturation, in chromosome
Protein separate with DNA (deoxyribonucleic acid) (DNA), thus discharge DNA.In addition, alkaline lysate, pH value model
Enclose 12～13.5, especially pH value range 12～12.6 the removable substantial amounts of keratin of lysate；Under strongly alkaline conditions,
Sodium lauryl sulphate can make protein precipitate.After fish cell is broken, ethylenediaminetetraacetic acid or ethylenediaminetetraacetic acid
Sodium salt can the metal ion such as magnesium ion in chela and Fish Tissue or cell and calcium ion, thus suppress deoxyribonuclease pair
The Degradation of DNA.Sodium chloride is conducive to improving DNA dissolubility in lysate, increases DNA stability, makes to be prone to DNA's
Preserve.
Discharge the principle of DNA based on above-mentioned cracking Fish Tissue, utilize the lysate that the present invention provides, can make
DNA in Fish Tissue quickly discharges, and DNA is not degraded, obtain rapidly quality and purity higher containing DNA's
Solution, lays the foundation for follow-up molecular biology research.
It should be noted that " Fish " described in the present invention defined in biotaxy are being: Fish be body by corneoscute,
Using gill respiration, with fin as organ of locomotion with the alternating temperature aquatic vertebrate that upper lower jaw is ingested, belong in Chordata
Vertebrate.
Further, described in present invention Fish Tissue can be the groups such as the muscle of Fish, internal organs, fin, scale and skeleton
Knit.Lysate disclosed in the embodiment of the present invention is applicable to the cracking of any tissue of Fish and released dna；Preferably, the flesh of Fish
Meat, internal organs, fin, scale and skeleton；It is further preferred that muscle, internal organs, fin and scale；The most preferably, muscle
And internal organs, wherein internal organs can be liver；Most preferably, muscle.
Further, highly basic described in the present invention refers to that alkali is dissolved in water and can occur full-ionized, 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 ethylenediaminetetraacetic acid
Disodium.
The following molar concentration containing EDTA to prepare 1L is as 2mM, and the molar concentration of NaCl is the quality of 1mM, SDS
Mark is 0.2%, as a example by the molar concentration of the hydroxide ion of NaOH is the lysate of 200mM, the lysate of the present invention is described
Preparation method:
The most accurately weighed 0.548g EDTA, 0.058g NaCl, 2.017g SDS, 7.999g NaOH are dissolved separately in
In distilled water, the solution containing these four solute is being mixed, is being settled to 1L with water,.
It will be appreciated by persons skilled in the art that in molecular biology research, the water used in reagent preparation is usually
Distilled water, distilled water, deionized water and ultra-pure water, be wherein excellent with ultra-pure water and deionized water.There is no the feelings of specified otherwise
Under condition, water used in embodiments of the invention all refers to distilled water, distilled water deionized water.
Lysate in the embodiment of the present invention can be preferably: ethylenediaminetetraacetic acid and/or disodium edta
Molar concentration is preferably 3～8mM；The molar concentration of sodium chloride is 3～7mM；The mass fraction of sodium lauryl sulphate be 0.5～
5%；The molar concentration of the hydroxide ion of highly basic is 150～250mM.The lysate of this concentration range not only can obtain high concentration
High-quality DNA, and in the solution containing DNA prepared, follow-up PCR reaction is had little to no effect by the reagent of residual.
Lysate in the embodiment of the present invention can also be more preferably: ethylenediaminetetraacetic 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 sodium lauryl sulphate is 1%；Highly basic
The molar concentration of hydroxide ion is 200mM.The lysate of this concentration not only can obtain the high-quality DNA of higher concentration, and
In the solution containing DNA prepared, follow-up PCR reaction is had little to no effect by the reagent of residual.
In a second aspect of the present invention, the embodiment of the present invention discloses again a kind of cleavage method, utilizes above-mentioned lysate to make
Fish Tissue cracks and discharges DNA, comprises the following steps: mixed with lysate by Fish Tissue, the water-bath of 20～85 DEG C
Middle holding 10～30min, centrifugal treating afterwards, it is thus achieved that supernatant be the solution containing DNA.
It can be seen that utilize lysate disclosed in first aspect present invention to extract in Fish Tissue from above-mentioned cleavage method
DNA, operating process is very simple, and the time is short, can realize the rapid extraction to Fish Tissue DNA.
It should be noted that before utilizing lysate to extract Fish Tissue DNA, first have to lysate is carried out at sterilizing
Reason.And the method that reagent carries out sterilization treatment is this area routine techniques means, research worker can be according to the bar of laboratory
Part selects voluntarily, and therefore, therefore not to repeat here for the present invention.If additionally, research worker is not to enter the supernatant obtained at once
Row subsequent experimental, can separate supernatant, save backup in-20 DEG C.Also it is ability for separating the method for supernatant
The customary means of field technique personnel, therefore not to repeat here for the present invention.
In the embodiment of cleavage method, water bath condition is preferably: keep 10～20min in the water-bath of 50～65 DEG C；
More preferably: in the water-bath of 55 DEG C, keep 10min.
In the embodiment of cleavage method, Fish Tissue can be preferably with the hybrid mode of lysate: vibration.Vibration side
Formula can be to realize with mini-vibrator, it is also possible to is artificial vibration, the most fully connects with lysate realizing Fish Tissue
Touch, make Fish Tissue and cell rapid disruption and released dna, thus realize DNA rapid extraction.Additionally, duration of oscillation can be
Tens seconds, such as 20 seconds, 40 seconds, generally need not be more than 60 seconds.Certainly those skilled in the art can according to practical experience voluntarily
Holding, the present invention is not especially limited at this.Centrifugal condition is also the means that those skilled in the art commonly use, and can be 10000g
Centrifugal 2～5min, it is also possible to more than 5000g is centrifuged more than 5min, it is proposed that need not be more than 10min, and the present invention does not make at this specifically
Limit.G described herein is that power about 9.8N, 10000g that acceleration produces are about the centrifugal force of 10000 kilograms.
In the embodiment of cleavage method, it is preferable that Fish Tissue is 10～25mg:200～400 with the ratio of lysate
μL；It is highly preferred that the ratio of Fish Tissue and lysate is 25mg:400 μ L.The addition 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 too much, also can reduce supernatant (containing DNA while causing the unnecessary waste of reagent
Solution) in DNA content, thus follow-up study is had a negative impact.
In a third aspect of the present invention, the embodiment of the invention also discloses a kind of two-component-type Fish Tissue DNA extraction liquid,
Including the above-mentioned lysate as the first component with as the neutralizer of second component, neutralizer is to include: containing tween 20
The Tris-HCl buffer that pH value is 6.4～7.4, wherein the mass fraction of the tween 20 in neutralizer (tween-20) is
0.02%～0.1%, the molar concentration of the Tris in neutralizer is 0.2～1mM.Wherein, for the composition of lysate, refer to
The related content of a kind of lysate that first aspect present invention is recorded, therefore not to repeat here for the present invention.
Narration according to a first aspect of the present invention, those skilled in the art are it is known that above-mentioned lysate is by fish
DNA in class loading discharges, and narration according to a second aspect of the present invention, those skilled in the art it will also be appreciated that
How to prepare the solution containing DNA.During biological study, preparation DNA is an element task, generally also needs to system
Standby DNA carries out follow-up research work, such as, carries out amplification in vitro to containing a certain specific DNA in DNA solution, the most logical
The most described polymerase chain reaction (PCR), the various materials in lysate are likely to affect PCR reaction, mainly pass through
The activity reducing archaeal dna polymerase affects PCR reaction.Tris-HCl in neutralizer provides a buffer system for DNA, at this
In individual system, DNA is steady statue, and Tris-HCl interacts with tween 20 can play the effect of protection archaeal dna polymerase, for
PCR reaction provides favourable guarantee.
In the embodiment of two-component-type Fish Tissue DNA extraction liquid, it is preferable that neutralizer is to include: containing tween 20
The Tris-HCl buffer that pH value is 6.8, wherein, the mass fraction of the tween 20 in neutralizer is 0.05%；In neutralizer
The molar concentration of Tris be 0.5mM.
In the embodiment of two-component-type Fish Tissue DNA extraction liquid, it is highly preferred that neutralizer also comprises bovine serum albumin
At least one in (BSA), gelatin and dithiothreitol, DTT (DTT) in vain.Bovine serum albumin (BSA), gelatin and dithiothreitol, DTT
(DTT) any one or the combination in, is more beneficial for protecting the DNA polymerase activity in PCR system, so that PCR reaction is suitable
Profit is carried out.
It should be noted that gelatin is the structure and relative molecular weight do not fixed, by animal skin, bone, sarolemma, flesh evil spirit
Degrade Deng the collagenous portion in connective tissue and become white or faint yellow, translucent, the thin slice of micro-strip gloss or powder.This
Gelatin used in bright embodiment is not edible or the other gelatin of technical grade, and applies to scientific research ground impurity content very
Low high-purity gelatin.
In the embodiment of two-component-type Fish Tissue DNA extraction liquid, bovine serum albumin (BSA), gelatin and two sulfur Soviet Union
Sugar alcohol (DTT) is more preferably: cumulative volume based on neutralizer, neutralizer also comprises the Ox blood serum egg of 0.02～0.1mg/mL
In vain, at least one in the gelatin of 0.2～2mg/mL and the dithiothreitol, DTT of 0.02～1mg/mL.
It will be appreciated by persons skilled in the art that and the solution containing DNA should reduce protein content as far as possible, and by sending out
The research of a person of good sense confirms, it is favourable for adding a small amount of BSA and/or gelatin in the solution containing DNA to PCR reaction.
In a fourth aspect of the present invention, embodiment of the invention discloses that one utilizes above-mentioned two-component-type Fish Tissue
DNA extraction liquid extracts the method for Fish Tissue DNA, comprises the following steps: mixed with above-mentioned lysate by Fish Tissue, 20
～the water-bath of 85 DEG C keeps 10～30min, then mix with above-mentioned neutralizer, centrifugal treating afterwards, it is thus achieved that supernatant be
Solution containing DNA.Wherein, for lysate and the composition of neutralizer, refer to the one pair that third aspect present invention is recorded
Related content in component type Fish Tissue DNA extraction liquid, therefore not to repeat here for the present invention.
For the hybrid mode of the ratio of Fish Tissue Yu lysate, Fish Tissue and lysate, Fish Tissue and cracking
The mixed water bath condition of liquid, those skilled in the art are referred in a kind of cleavage method that second aspect present invention is recorded
Related content, therefore not to repeat here for the present invention.
In extracting the embodiment of method of Fish Tissue DNA, it is preferable that the volume ratio of lysate and neutralizer is 10:3
～6.This parameter not only can neutralize the alkalescence of the solution containing DNA, moreover it is possible to gives archaeal dna polymerase with strong protection, makes PCR anti-
Should be smoothed out.
In extracting the embodiment of method of 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.
Need it is further noted that the hybrid mode of Fish Tissue and lysate can be vibration, neutralizer and water-bath
After lysate and the hybrid mode of mixture of Fish Tissue can be vibration.Can join for mode of oscillation and duration of oscillation
Examining the related content in the embodiment that second aspect present invention is recorded, therefore not to repeat here for the present invention.Permissible for centrifugal condition
With reference to the related content in the embodiment that second aspect present invention is recorded, therefore not to repeat here for the present invention.
In a fifth aspect of the present invention, embodiment of the invention discloses that a kind of Fish Tissue DNA extraction kit, including
Above-mentioned lysate and above-mentioned neutralizer.For lysate and the composition of neutralizer, refer to third aspect present invention and record
A kind of two-component-type Fish Tissue DNA extraction liquid in related content, therefore not to repeat here for the present invention.
In the embodiment of the Fish Tissue DNA extraction kit of the present invention, a kind of Fish Tissue DNA extraction kit is also
Comprise Fish Tissue DNA extraction operating instruction.For the operating procedure described in Fish Tissue DNA extraction operating instruction,
The one that refer to fourth aspect present invention record utilizes above-mentioned two-component-type Fish Tissue DNA extraction liquid to extract Fish Tissue
The method of DNA, therefore not to repeat here for the present invention.
For example, Fish Tissue DNA extraction operating instruction can be: is washed with deionized water only by Fish Tissue, takes
25mg Fish Tissue, after shredding, puts in centrifuge tube, adds the lysate of 400 μ L, vibrates 20 seconds, afterwards the water-bath of 55 DEG C
Middle holding 10min, adds 120 μ L neutralizers, vibrates 20 seconds, and last 10000g is centrifuged 2min, takes supernatant, be containing
The solution of DNA.
In a sixth aspect of the present invention, embodiment of the invention discloses that above-mentioned Fish Tissue DNA extraction kit is carrying
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 Ping.Such as, quickly detection, the qualification etc. of fish species of Fish genomes.The research of Fish
Personnel can need to select voluntarily according to research, and the present invention is numerous to list herein.
In a seventh aspect of the present invention, embodiment of the invention discloses that a kind of PCR system expanding Fish Tissue DNA,
This PCR system includes the solution containing DNA obtained by the method by said extracted Fish Tissue DNA, based on this PCR system
Cumulative volume, the volume fraction of the solution containing DNA is 2%～15%.This PCR system can be:
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 accurately weighed described in the present invention 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 reagent；
Taq archaeal dna polymerase originates from Thermo Fischer Scient Inc. (Thermo Fisher Scientific)；
DNAMarker is Shanghai Sheng Gong Bioisystech Co., Ltd product.
Test material is the muscle of Carnis Pseudosciaenae (Larimichthys polyactis), liver, middle body vertebrae, squama
Sheet and fin.
18S rRNA gene primer sequence stems from list of references: 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.
CO I gene primer sequence stems from list of references: 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 primer sequence and CO I gene primer sequence 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 of the U.S.；
Gel imaging system, Liuyi Instruments Plant, Beijing.
Embodiment A1～A4, embodiment B1～B4 and embodiment C1～C4
The extraction of the DNA in Carnis Pseudosciaenae tissue.Wherein used Carnis Pseudosciaenae tissue is respectively scale, skeleton, muscle, liver
Dirty.
Embodiment A1～A4
Clean Carnis Pseudosciaenae tissue with the distilled water of sterilizing, take the Carnis Pseudosciaenae tissue after 25mg cleans, after shredding, put into 1.5mL
In centrifuge tube, add the lysate of 400 μ L, vibrate 20 seconds, the water-bath of 55 DEG C keeps 10min, add 120 μ L and neutralize
Liquid, vibrates 20 seconds, and then 10000g is centrifuged 2min, takes supernatant, obtains the solution containing DNA.
The reaction condition of embodiment A1～A4 and embodiment B1～B4 and embodiment C1～C4 differ only in lysate
Different with the respective reagent proportioning in neutralizer, it is shown in Table 1:
The composition of reagent in table 1 lysate and neutralizer
Numeral in embodiment A1～A4, embodiment B1～B4 and embodiment C1～C4 represents the different tissues of Carnis Pseudosciaenae,
Wherein 1, scale, 2, skeleton, 3, muscle, 4, liver.
It is dense that the solution containing DNA preparing embodiment A1～A4, embodiment B1～B4 and embodiment C1～C4 carries out DNA
Degree measures, and the results are shown in Table 2:
Table 2 DNA concentration
From Table 2, it can be seen that for extraction used in same tissue, embodiment B1～B4 and embodiment C1～C4
In solution containing DNA prepared by liquid, DNA concentration is without prepared by extracting solution used in significant difference, embodiment A1～A4 containing
DNA concentration in the solution of DNA is had to be significantly lower than the former two.
The solution containing DNA prepared using embodiment A1～A4, embodiment B1～B4 and embodiment C1～C4 is as DNA mould
Plate solution, expands Eukaryotic 18S rRNA gene, it is contemplated that amplified production be 140bp, 18S rRNA gene primer sequence such as
Under:
Forward primer: 5'-TCTGCCCTATCAACTTTCGATGG-3'
Downstream primer: 5'-TAATTTGCGCGCCTGCTG-3'
PCR system cumulative volume is 20 μ L, and it is as shown in table 3 below that it contains reagent:
Each reagent dosage and final concentration in table 3 18S rRNA gene PCR system
PCR response procedures is: 95 DEG C of degeneration 5min；95 DEG C of degeneration 30s, 59 DEG C of annealing 30s, 72 DEG C extend 30s is one
Circulation, 30 circulations altogether；Then 72 DEG C keep 10min.Reaction is cooled to 4 DEG C after terminating.
The each 6 μ L of PCR primer detect through 2% agarose gel electrophoresis, and nucleic acid dye Gel Red dyes, gel imaging system
Analyze, result (M in Fig. 1 represents DNAMarker) as shown in Figure 1.As shown in Figure 1, product size is consistent with expection 140bp,
Illustrate that amplification has obtained 18S rRNA gene outcome.Electrophoresis result can be as the basic foundation of molecular biological analysis.From little
The DNA that the different tissues of Channa argus extracts carries out PCR, and from the point of view of electrophoresis result, the effect of skeleton, scale and fin is than muscle and liver
Slightly worse.DNA extraction effect in muscle and liver organization is preferable, the two no significant difference.It is to say, the fish that the present invention provides
Class loading DNA extraction liquid and extracting method are capable of the DNA extraction to Fish different tissues, and the extraction to muscle and liver is imitated
Fruit is better than the extraction effect to skeleton, scale and fin.
From the point of view of result in conjunction with table 2 and Fig. 1, for the same tissue of Carnis Pseudosciaenae, embodiment B1～B4 and embodiment C1～
In solution containing DNA prepared by extracting solution used in C4, DNA concentration is without significant difference, and identical at PCR reaction condition
In the case of, the electrophoresis result of PCR primer but shows that the DNA solution prepared with embodiment B1～B4 is carried out for DNA profiling solution
The PCR primer amount that PCR obtains is considerably less than embodiment C1～C4, traces it to its cause, it may be possible to used in embodiment B1～B4
In extracting solution, each reagent content is high, it is suppressed that PCR reacts.
Embodiment D～embodiment S
Embodiment D～embodiment S are same with the Fish Tissue DNA extraction liquid phase used in embodiment C1～C4, and difference is
The consumption of Carnis Pseudosciaenae tissue types, Carnis Pseudosciaenae tissue, lysate and neutralizer, and water bath condition is different.
| Embodiment | Tissue types | Tissue (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 | Skeleton | 25 | 400 | 120 | 55 | 10 |
| L | Skeleton | 10 | 200 | 60 | 50 | 20 |
| M | Skeleton | 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 |
The solution containing DNA preparing embodiment D～embodiment S carries out DNA concentration mensuration, and inventor finds, processes
DNA concentration in the solution containing DNA that muscle and liver obtain is 50～200ng/ μ L, process that fin obtains containing DNA
Solution in the concentration of DNA be 20～100ng/ μ L, process the concentration of DNA in the solution containing DNA that scale obtains 8
～40ng/ μ L.And A in the above-mentioned solution containing DNA260/A280Ratio all between 1.2～1.6, the reality of the present invention is described
The DNA purity executing example acquisition is good.
Comparative example
Phenol-chloroform extraction method is utilized to extract the DNA in Carnis Pseudosciaenae muscle.Wherein, phenol-chloroform extraction method sees " molecule gram
Grand experiment guide " third edition, the first volume: 463-485.
The DNA that in the Carnis Pseudosciaenae muscle that phenol-chloroform extraction method will be utilized to extract, DNA and embodiment C1～C4 obtain enters simultaneously
Performing PCR is tested, and verifies the extracting method of the Fish Tissue DNA of the present invention.
The Mitochondrial cytochrome c oxidase I (cytochrome coxidase I, CO I) of amplification Carnis Pseudosciaenae DNA bar code
Gene, a length of 658bp, its primer sequence is as follows:
CO I upstream region of gene primer: 5'-TCAACCAACCACAAAGACATTGGCAC-3'
CO I downstream of gene primer: 5'-TAGACTTCTGGGTGGCCAAAGAATCA-3'
PCR system cumulative 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
The 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 adds water and is supplemented to 20 μ L.
CO I gene PCR response procedures is: 95 DEG C of degeneration 5min；95 DEG C of degeneration 30sec, 55 DEG C of annealing 35sec, 72 DEG C are prolonged
Stretching 35sec is a circulation, altogether 30 circulations；Then 72 DEG C keep 10min.4 DEG C it are cooled to after end.
The each 6 μ L of PCR primer detect through 2% agarose gel electrophoresis, and nucleic acid dye Gel Red dyes, and gel systems is taken pictures,
(1,2,3 and 4 in Fig. 2 represent with muscle, liver, scale and the skeleton of Carnis Pseudosciaenae for sample acquisition result respectively as shown in Figure 2
PCR primer, 5 represent utilize phenol-chloroform extraction method process Carnis Pseudosciaenae muscle samples obtain PCR primer, M represents
DNAMarker).As shown in Figure 2, the muscle of process, liver, scale and bone samples all obtain the special of expection size 658bp
Property band, the DNA extraction effect in muscle, liver, scale is essentially identical with the DNA utilizing phenol-chloroform to extract, the effect of skeleton
Slightly worse.The quality of the DNA illustrating to utilize the method for the extraction Fish Tissue DNA of the present invention to obtain and content all no less than phenol-
The DNA that chloroform extraction obtains.Moreover, relative to phenol-chloroform extraction method, the Fish Tissue DNA extraction method of the present invention
Simple to operate, the shortest, the toxicity of extracting solution is low.Additionally, utilize Fish Tissue DNA extraction liquid and the extracting method system of the present invention
The gene directly expanding long segment for the DNA profiling solution gone out also has good effect.
It should be noted that Canada scientist Hebert proposed DNA bar code technology in 2003, in the same year, CO is proposed
I gene is as bar code target.DNA bar code technology selects one section of species specificity sequence as the mark of species identification, CO I
Gene, from proposing as target application so far, has played important function, especially in animals and plants and microbial identification and species identification
It demonstrates unrivaled superiority in the qualification of Fish, is that Fish identify most potential instrument, and goes back in the world
Having the data base of special fish DNA bar code, the Rapid identification for Fish provides important support.
Technical scheme 1
A kind of lysate, it is used for cracking Fish Tissue and discharges DNA, it is characterised in that including:
Ethylenediaminetetraacetic acid and/or disodium edta, sodium chloride, sodium lauryl sulphate, highly basic and water,
Wherein for lysate, the molar concentration of ethylenediaminetetraacetic acid and/or disodium edta be 2～
10mM, the molar concentration of sodium chloride is 1～10mM, and the mass fraction of sodium lauryl sulphate is 0.1～10%, and highly basic
The molar concentration of hydroxide ion is 100～300mM.
Technical scheme 2
According to the lysate described in technical scheme 1, it is characterised in that
The molar concentration of described ethylenediaminetetraacetic acid and/or disodium edta is 3～8mM；Described sodium chloride
Molar concentration is 3～7mM；The mass fraction of described sodium lauryl sulphate is 0.5～5%；The hydroxide ion of described highly basic
Molar concentration be 150～250mM.
Technical scheme 3
According to the lysate described in technical scheme 1, it is characterised in that
The molar concentration of described ethylenediaminetetraacetic acid and/or disodium edta is 5mM；Described sodium chloride mole
Concentration is 5mM；The mass fraction of described sodium lauryl sulphate is 1%；The molar concentration of the hydroxide ion of described highly basic is
200mM。
Technical scheme 4
According to the lysate described in any one of technical scheme 1 to 3, it is characterised in that described highly basic selected from NaOH and/or
KOH。
Technical scheme 5
A kind of two-component-type Fish Tissue DNA extraction liquid, it is characterised in that include the technical scheme 1 as the first component
～4 lysate described in any one and the neutralizer as second component, described neutralizer is to include: containing the pH of tween 20
Value is the Tris-HCl buffer of 6.4～7.4, the mass fraction of the tween 20 in wherein said neutralizer be 0.02%～
0.1%, the molar concentration of the Tris in described neutralizer is 0.2～1mM.
Technical scheme 6
According to the DNA extraction liquid described in technical scheme 5, it is characterised in that described neutralizer is to include: containing tween 20
The Tris-HCl buffer that pH value is 6.8, the mass fraction of the tween 20 in wherein said neutralizer is 0.05%；Described
The molar concentration of the Tris in neutralizer is 0.5mM.
Technical scheme 7
According to the DNA extraction liquid described in technical scheme 5 or 6, it is characterised in that described neutralizer also comprises in following material
At least one:
Bovine serum albumin,
Gelatin, and
Dithiothreitol, DTT.
Technical scheme 8
According to the DNA extraction liquid described in technical scheme 7, it is characterised in that cumulative volume based on described neutralizer, described in
The gelatin of bovine serum albumin, 0.2～the 2mg/mL of 0.02～0.1mg/mL and the two sulfur Soviet Unions of 0.02～1mg/mL are also comprised with liquid
At least one in sugar alcohol.
Technical scheme 9
A kind of cleavage method, it utilizes the lysate as described in any one of technical scheme 1 to 4 to make Fish Tissue discharge
DNA, it is characterised in that comprise the following steps:
Fish Tissue is mixed with lysate, holding 10～30min in the water-bath of 20～85 DEG C, centrifugal treating afterwards,
The supernatant obtained is the solution comprising DNA.
Technical scheme 10
According to the method described in technical scheme 9, it is characterised in that keep 10～20min in the water-bath of 50～65 DEG C.
Technical scheme 11
According to the method described in technical scheme 10, it is characterised in that keep 10min in the water-bath of 55 DEG C.
Technical scheme 12
According to the method described in technical scheme 9, it is characterised in that the mode of described mixing is vibration.
Technical scheme 13
According to the method described in technical scheme 9, it is characterised in that described Fish Tissue is 10 with the ratio of described lysate
～25mg:200～400 μ L.
Technical scheme 14
According to the method described in technical scheme 13, it is characterised in that described Fish Tissue with the ratio of described lysate is
25mg:400μL。
Technical scheme 15
A kind of method extracting Fish Tissue DNA, it is characterised in that comprise the following steps:
Fish Tissue is mixed with the lysate described in any one of technical scheme 5 to 8, keeps in the water-bath of 20～85 DEG C
10～30min, then mix with the neutralizer described in any one of technical scheme 5 to 8, centrifugal treating afterwards, it is thus achieved that supernatant i.e.
For the solution containing DNA.
Technical scheme 16
According to the method described in technical scheme 15, it is characterised in that keep 10～20min in the water-bath of 50～65 DEG C.
Technical scheme 17
According to the method described in technical scheme 16, it is characterised in that keep 10min in the water-bath of 55 DEG C.
Technical scheme 18
According to the method described in any one of technical scheme 15 to 17, it is characterised in that described lysate and described neutralizer
Volume ratio be 10:3～6.
Technical scheme 19
According to the method described in technical scheme 18, described Fish Tissue is 10～25mg:200 with the ratio of described lysate
～400 μ l.
Technical scheme 20
According to the method described in technical scheme 19, it is characterised in that described Fish Tissue, described first component and described
The ratio of two components is 25mg:400 μ L:120 μ L.
Technical scheme 21
According to the method described in technical scheme 15, it is characterised in that the mode of described mixing is vibration.
Technical scheme 22
According to the method described in technical scheme 15, it is characterised in that described Fish Tissue is selected from: muscle, internal organs, fin,
Scale or skeleton.
Technical scheme 23
A kind of Fish Tissue DNA extraction kit, it is characterised in that include the cracking described in any one of technical scheme 1 to 4
Neutralizer described in liquid and any one of technical scheme 5 to 8.
Technical scheme 24
According to the Fish Tissue DNA extraction kit described in technical scheme 23, it is characterised in that also comprise Fish Tissue
DNA extraction operating instruction.
Technical scheme 25
The application in extracting Fish Tissue DNA of the test kit described in technical scheme 23 or 24.
Technical scheme 26
A kind of PCR system expanding Fish Tissue DNA, it is characterised in that this PCR system includes by technical scheme 15 to 22
The solution containing DNA obtained by method described in any one, cumulative volume based on this PCR system, the described solution containing DNA
Volume fraction is 2%～15%.
Technical scheme 27
According to the PCR system described in technical scheme 26, it is characterised in that cumulative volume based on this PCR system, described contain
The volume fraction of the solution of DNA is 10%.
The above is for only for ease of those skilled in the art and understands technical scheme, not in order to limit
The present invention.All within the spirit and principles in the present invention, any modification, equivalent substitution and improvement etc. made, should be included in this
Within the protection domain of invention.
Claims (10)
1. a lysate, it is used for cracking Fish Tissue and discharges DNA, it is characterised in that including:
Ethylenediaminetetraacetic acid and/or disodium edta, sodium chloride, sodium lauryl sulphate, highly basic and water,
Wherein for lysate, the molar concentration of ethylenediaminetetraacetic acid and/or disodium edta be 2～
10mM, the molar concentration of sodium chloride is 1～10mM, and the mass fraction of sodium lauryl sulphate is 0.1～10%, and highly basic
The molar concentration of hydroxide ion is 100～300mM.
Lysate the most according to claim 1, it is characterised in that
The molar concentration of described ethylenediaminetetraacetic acid and/or disodium edta is 3～8mM；Described sodium chloride mole
Concentration is 3～7mM；The mass fraction of described sodium lauryl sulphate is 0.5～5%；Rubbing of the hydroxide ion of described highly basic
Your concentration is 150～250mM.
Lysate the most according to claim 1, it is characterised in that
The molar concentration of described ethylenediaminetetraacetic acid and/or disodium edta is 5mM；The molar concentration of described sodium chloride
For 5mM；The mass fraction of described sodium lauryl sulphate is 1%；The molar concentration of the hydroxide ion of described highly basic is
200mM。
4. according to the lysate described in any one of claims 1 to 3, it is characterised in that described highly basic is selected from NaOH and/or KOH.
5. a two-component-type Fish Tissue DNA extraction liquid, it is characterised in that include the Claims 1 to 4 as the first component
Lysate described in any one and the neutralizer as second component, described neutralizer is to include: the pH value containing tween 20 is
The Tris-HCl buffer of 6.4～7.4, the mass fraction of the tween 20 in wherein said neutralizer is 0.02%～0.1%,
The molar concentration of the Tris in described neutralizer is 0.2～1mM.
DNA extraction liquid the most according to claim 5, it is characterised in that described neutralizer includes: containing tween 20
PH value is the Tris-HCl buffer of 6.8, and the mass fraction of the tween 20 in wherein said neutralizer is 0.05%；In described
It is 0.5mM with the molar concentration of the Tris in liquid.
7. according to the DNA extraction liquid described in claim 5 or 6, it is characterised in that described neutralizer also comprises in following material
At least one:
Bovine serum albumin,
Gelatin, and
Dithiothreitol, DTT.
DNA extraction liquid the most according to claim 7, it is characterised in that cumulative volume based on described neutralizer, described neutralization
Liquid also comprises the gelatin of bovine serum albumin, 0.2～the 2mg/mL of 0.02～0.1mg/mL and the two sulfur threoses of 0.02～1mg/mL
At least one in alcohol.
9. a cleavage method, it utilizes the lysate as described in any one of Claims 1-4 to make Fish Tissue discharge
DNA, it is characterised in that comprise the following steps:
Fish Tissue is mixed with lysate, the water-bath of 20～85 DEG C keeps 10～30min, centrifugal treating afterwards, it is thus achieved that
Supernatant be the solution comprising DNA.
Method the most according to claim 9, it is characterised in that keep 10～20min in the water-bath of 50～65 DEG C.
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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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