CN107447007A - A kind of DNA class loadings Sample storage liquid and application - Google Patents
A kind of DNA class loadings Sample storage liquid and application Download PDFInfo
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Abstract
The invention discloses a kind of DNA class loadings Sample storage liquid and application, belong to biological technical field, preserve in liquid, ammonium oxalate saturated solution, 0.05~2mM of disodium ethylene diamine tetraacetate, 10~50mM of trihydroxy aminomethane, Triton X-100 0.2~0.5%, sodium chloride 0.5~1.5%, pH value are controlled between 7.2~8.1.Preserve liquid composition it is simple, safe and nontoxic, inexpensive, chemical property is stable, it is not volatile, once preserve after do not have to the later stage supplement preservation liquid；Preserve liquid does not have dissection to DNA, can extract the DNA of large fragment；Extra process that it goes without doing when later stage DNA is extracted, DNA extractions are simple to operate；Tissue dewatering can be made by preserving liquid hypersaline environment, and " the loose shortcakeization " of tissue will not also be caused by preserving tissue even if long-time, therefore tissue will not be made to tail off.
Description
Technical field
The invention belongs to biological technical field, is related to the fixation and preservation of biological tissue, and in particular to a kind of DNA class loadings
Sample storage liquid and application.
Background technology
The permanent preservation of animal tissue is to carry out the important prerequisite of DNA sequencing, molecule genetics research and forensic identification etc.,
The quality that animal tissue preserves directly affects the yield and quality of later stage DNA extractions.Currently used animal tissue's store method
There are a low-temperature freezing, formaldehyde infusion method, paraffin imbedding and ethanol infusion method etc., different store methods have the characteristics of respective and excellent
Shortcoming.
Low-temperature freezing is suppressed using low temperature (in -20 DEG C of general refrigerators, -80 DEG C of ultra low temperature freezers or -196 DEG C of liquid nitrogen)
The activity of DNA enzymatic so that preserve tissue DNA for a long time, and this method preservation effect is preferable, but its preservation condition is strict, preserve cost
It is high and easily influenceed by the incident such as have a power failure.
Formaldehyde infusion method makes DNA enzymatic inactivate so as to preserve DNA by formaldehyde, and this method has preferably for animal tissue
Fixation.But formaldehyde can make protein that serious crosslinking occur with DNA, and it is difficult to be discharged completely from tissue to make DNA, separately
Outside, formaldehyde can also cause fragmentation and the modification of genomic DNA, cause to be difficult in later stage DNA extraction process to obtain a large amount of complete
Fragment.In addition, formaldehyde has very strong toxicity, this method has potentially hazardous to operating personnel.
Paraffin imbedding is also the conventional method that animal sample preserves, but its waxdip, embedding and storage process all can be right
DNA in animal tissue causes expendable damage.This method is also required to use formaldehyde, therefore will also result in later stage DNA and carry
The difficulty taken.
Animal tissue is dehydrated by ethanol infusion method using the osmosis of ethanol, DNA enzymatic inactivates and then preserves tissue DNA.
Although ethanol is rapid and nontoxic to tissue infiltration effect, there are some researches show preserve animal groups in ethanol for a long time
Knit can soft disperse, the tissue block for causing to preserve tails off, and ethanol also can produce dissection to genomic DNA, formed length compared with
Small DNA fragmentation, and ethanol highly volatile, concentration of alcohol can reduce after long-term preservation, be unable to reach good preservation effect.
The fields such as DNA sequencing, molecule genetics research and forensic identification are required to extract from the animal tissue of preservation
A large amount of and more complete DNA, but the quantity and matter for the DNA for extracting and purifying in the animal tissue preserved from above-mentioned conventional method
Amount is not often considerable, it is impossible to meets the needs of modern molecular biology research.Therefore, researcher in this field is directed to out
The store method of animal tissue can completely be preserved by sending out new.The guarantor of many cases animal tissue/cell is also disclosed that in the prior art
Deposit method.
As CN101668854A discloses composition, the system and method for preservation and/stabilized cells/macromolecular, its group
Compound includes chelating agent, chelating agent enhancing component and base, and coupled system can be such that cell preserves at ambient conditions and stably
Change.
CN103820320A discloses a kind of non-jelly type RNA protections liquid, by polyethyleneimine, ammonium chloride, ethylenediamine tetrem
Acid and its disodium salt, ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA) composition, can preserve biological sample (tissue, animal at room temperature
Tissue, plant tissue, bacterium, fungi, culture cell and blood cell) 14-42 days, period RNA keeps high integrity.
CN104630207A discloses a kind of marine animal Saving specimen and the integrated reagent of DNA extractions, including pH8.0
Tris, EDTA, SDS, Proteinase K, for the operation integrated with DNA extractions of marine animal Saving specimen.
CN105524916A discloses a kind of DNA classes Sample preservation dilution and its preparation, and it preserves liquid mainly by diformazan
Base sulfoxide, sorbierite, Tris-HCl, ethylenediamine tetra-acetic acid, Triton-100, NP-40, Chelex-100, Sodium azide, streptomysin
Formed with deionized purified water, after immersing DNA classes Sample Dilution preservation liquid, DNA can be with intact 37 in fresh tissue cells
Preserved 1 week at DEG C, preserve at 25 DEG C and preserved 6 months at 1 month, 4 DEG C, preserved for a long time at -20 DEG C or -80 DEG C.
But the existing store method still suffers from problems, tissue is such as preserved for a long time and causes the " loose crisp of tissue
Change " tissue is tailed off, though or DNA can be preserved can not extract the DNA of large fragment, or be needed during preserving it is more
Though secondary replacing or supplement preserve liquid and can realize Sample storage but extract the problems such as unfavorable for follow-up DNA.Therefore, develop
A kind of store method that is new, can completely preserving animal tissue is significant.
The content of the invention
In view of the above-mentioned problems of the prior art, the present invention is analyzed and studied for the preservation of animal tissue, open
The preservation liquid for sending out a kind of available for many animals tissue preserration.
An object of the present invention is to provide a kind of DNA class loadings Sample storage liquid.The tissue sample preserve liquid in
Under room temperature condition can long-term full preserve animal tissue sample, preserve its DNA and can extract the DNA of large fragment high quality.
The second object of the present invention is the preparation method for providing above-mentioned DNA class loadings Sample storage liquid.Pass through its preparation
Method, can it is easy, quickly prepare DNA class loading Sample storage liquid, it is easy to use.
The third object of the present invention is to provide the DNA applications of class loading sample, includes the preservation side of DNA class loading samples
Method that method, DNA class loading Sample storages kit, the DNA molecular based on DNA class loading Sample storage liquid detect etc..
For achieving the above object, specifically the present invention relates to following technical scheme：
First, the invention discloses a kind of DNA class loadings Sample storage liquid, its composition is by ammonium oxalate [(NH4)2C2O4], three
Hydroxyl amino methane [Tris], disodium ethylene diamine tetraacetate [EDTA-Na2.2H2O], sodium chloride [NaCl], polyethylene glycol octyl group benzene
Base ether [Triton X-100] and deionized purified water composition.
Preferably, in DNA class loadings Sample storage liquid, ammonium oxalate saturated solution, disodium ethylene diamine tetraacetate 0.05~
2mM, trihydroxy 10~50mM of aminomethane, Triton X-100 0.2~0.5% (v/v), sodium chloride 0.5~1.5%
(w/w), pH value control is between 7.2~8.1.
It is more highly preferred to, in DNA class loading Sample storage liquid, (NH4)2C2O460 DEG C of saturated solutions, Tris 0.24%
(w/v)、EDTA-Na2.2H2O 0.074% (w/v), NaCl 0.9% (w/v), Triton X-100 0.3% (v/v) and go from
Sub- purified water composition.
Secondly, the invention discloses a kind of preparation method of DNA class loadings Sample storage liquid, comprise the following steps：
(1) deionized water of certain volume is measured, is heated to 50 DEG C -60 DEG C, adds 80~140g (NH4)2C2O4, according to quantity
Take Tris, EDTA-Na2.2H2O, NaCl, Triton X-100, fully mix.
(2) solution adjusts pH value to 7.2~8.1, is settled to 1.0L.
(3) autoclaving packing is standby.
Preferably, a kind of preparation method of DNA class loadings Sample storage liquid, comprises the following steps：
(1) 900mL deionized waters are measured, are heated to 60 DEG C, sequentially add 140g (NH4)2C2O4、2.42g Tris、
0.74g EDTA-Na2.2H2O, 9.0g NaCl, 3.0mL Triton X-100, are sufficiently stirred mixing.
(2) pH value is adjusted to 7.5 with NaOH solution, be settled to 1.0L.
(3) 115 DEG C of sterilizing 20min of high pressure, are dispensed standby immediately.
In addition, the invention discloses the application of above-mentioned DNA class loadings Sample storage liquid, the application includes DNA sequencing, divided
Application in sub- genetics research and forensic identification.
Based on above-mentioned application, the invention discloses a kind of store method of DNA class loadings sample, including above-mentioned preservation is used
The step of liquid preserves to tissue sample.
The invention also discloses DNA class loading Sample storage kits, including above-mentioned preservation liquid, sterile storage containers.
Tissue sample is carried out the invention also discloses a kind of method of DNA molecular detection, including using above-mentioned preservation liquid
The step of preservation.
Preferably, the step of also including DNA extractions in the method for DNA molecular detection, further also using extraction
DNA carry out molecular Biological Detection the step of.
The present invention achieves following beneficial effect：
(1) present invention preserve liquid composition it is simple, safe and nontoxic, inexpensive, chemical property is stable, it is not volatile, once protect
Do not have to later stage supplement after depositing and preserve liquid；
(2) present invention preserves liquid does not have dissection to DNA, can extract the DNA of large fragment；
(3) tissue of liquid preservation, extra process that it goes without doing when later stage DNA is extracted, DNA extraction behaviour are preserved using the present invention
Make simple；
(4) preservation liquid hypersaline environment of the present invention can make tissue dewatering, and preserving tissue even if long-time will not also cause
" the loose shortcakeization " of tissue, therefore tissue will not be made to tail off.
Brief description of the drawings
It is respectively the Mammalia preserved at ambient temperature with the preservation liquid prepared by absolute ethyl alcohol and embodiment 1 that Fig. 1, which is,
(Mammalia) rabbit (Oryctolagus cuniculus), Yi guiding principles (Echiurida) Urechis uniconctus (Urechis
Unicinctus), Crustachia (Crustacean) Penaeus Vannmei (Penaeus vannamei) and Bivalvia (Bivalvia)
The agarose for the genomic DNA that the musculature of Ruditapes philippinarum (Ruditapes philippinarum) was extracted after 50 days
Gel electrophoresis figure；M representation DNAs Marker in figure (stripe size is respectively 5000,3000,2000,1000,750 from top to bottom,
500,250,100bp)；1 is the rabbit organization preserved with absolute ethyl alcohol, and 2 be with the family for preserving liquid and preserving prepared by embodiment 1
Rabbit is organized；3 be the Urechis uniconctus tissue preserved with absolute ethyl alcohol, and 4 be with the monocyclic thorn for preserving liquid and preserving prepared by embodiment 1
Yi is organized；5 be the Penaeus Vannmei tissue preserved with absolute ethyl alcohol, and 6 be with the South America for preserving liquid and preserving prepared by embodiment 1
White shrimp is organized；7 be the Ruditapes philippinarum tissue preserved with absolute ethyl alcohol, and 8 be to preserve what liquid preserved with prepared by embodiment 1
Ruditapes philippinarum tissue.
Embodiment
It is noted that described further below is all exemplary, it is intended to provides further instruction to the present invention.It is unless another
Indicate, all technologies used herein and scientific terminology are with usual with general technical staff of the technical field of the invention
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe embodiment, and be not intended to restricted root
According to the illustrative embodiments of the present invention.As used herein, unless the context clearly indicates otherwise, otherwise singulative
It is also intended to include plural form, additionally, it should be understood that, when in this manual using term "comprising" and/or " bag
Include " when, it indicates existing characteristics, step, operation and/or combinations thereof.
Herein, term " DNA class loadings sample " refers to that needs extract biological tissue's sample of large fragment DNA from tissue
Product.
Term " large fragment DNA ", refer to that more complete large fragment genomic DNA can be obtained from tissue sample, such as
The high quality DNA of 23kb fragments.
Really as described in the background art, the existing store method still suffers from problems, such as preserves tissue for a long time
Causing " loose shortcakeization " of tissue makes tissue tail off, though or DNA can be preserved can not extract the DNA of large fragment, or
Though be preserve during need repeatedly change or supplement preserve liquid and can realize Sample storage but for follow-up DNA extraction not
The problems such as sharp.Therefore, the present invention develops a kind of store method that is new, can completely preserving animal tissue emphatically.
A kind of DNA class loadings Sample storage liquid is disclosed in an embodiment of the invention, its composition is by ammonium oxalate, three
Hydroxyl amino methane, disodium ethylene diamine tetraacetate, sodium chloride, Triton X-100 and deionized purified water composition.
The main function that the present invention preserves each component in liquid is as follows：
The main reason for suppressing DNA enzymatic activity, causing DNA degradation is dissection of the DNA enzymatic to DNA, and DNA enzymatic is active
Performance need Mg2+Presence (Mg2+It is the confactor of DNA enzymatic), EDTA is a kind of strong metallic divalent cations chela
Mixture, therefore the present invention mainly chelates the Mg in animal tissue cell with EDTA2+To suppress the activity of DNA enzymatic；
Weakly alkaline environment contributes to DNA stabilization, and the alkalescence of Tris alkali is stronger, is commonly used for by acid to a wide range of of alkalescence
The buffer solution of pH value (adds EDTA such as in Tris-HCl buffer solutions and TE buffer solutions is made；It is if the hydrochloric acid for adjusting pH value is molten
Liquid changes acetic acid into, then obtains TAE buffer solutions, change boric acid into and then obtain tbe buffer liquid, both buffer solutions are generally used for nov nucleic acid
The buffer solution of swimming), it is of the invention then made using Tris between preservation liquid pH value maintains 7.2~8.1, to strengthen DNA in tissue
Stability；
Saturated oxalic acid ammonium salt solution can suppress the growth of microorganism, on the one hand the growth of microorganism can reduce the pH for preserving liquid
Value, the stability of DNA systems is destroyed, the metabolite of microorganism also results in animal tissue and rotted in addition, so as to cause DNA's
Degraded；It is the growth for suppressing microorganism that the present invention, which uses the ammonium oxalate solution one under saturation state,；Second, can with it is a variety of
Metal ion into salt slightly soluble (as and Mg2+Etc. the oxalates that reaction product is slightly soluble in water, Mg is further removed2+Deng suppression DNA drops
Solve the activity of enzyme)；In addition, saturated solution is advantageous to the partially dehydrated sizing of animal tissue, prevent that tissue is loose；The present invention is preserving
In the process for preparation of liquid, found in preliminary experiment scheme, although antibiotic agents (such as penicillin, streptomysin) can effectively press down
Bacterium, but it is chronically at unstable under room temperature condition, preserving the liquid later stage is changed into muddiness and can not extract DNA (even if being stored in 4 DEG C of conditions
Under, do not change persistently and preserve liquid, the antibacterial resistance of its antibiotic can also disappear after one month), the final non-selected antibiosis of the present invention
The use of element, and use part that there is the saturated oxalic acid ammonium high level salt solution of fungistatic effect, fungistatic effect can be effectively played.
The DNA of animal tissue's sample is easy to degrade, and more than certain time, fresh sample DNA degradation is serious, existing
No matter 95% ethanol immersion or formaldehyde immersion, the DNA degradation of extraction is all than more serious, comparatively, cryopreservation and newly
The effect of fresh sample is preferable；The present invention is directed to the preservation of animal tissue's sample, using Triton X-100 (Triton
X-100 membrane passage) is increased, the ion for helping to preserve in liquid enters cell, can be with more efficiently maintenance DNA's
It is stable；Also be effectively reduced the usage amount of preservation liquid simultaneously (need to ensure enough ethanol during usually ethanol fixation preservation sample
Dosage, if field sampling do not have sufficiently large container be used for it is fixed preserve sample, often using the side of repeatedly renewal preservation liquid
Method)；
Sodium chloride solution of the present invention has the function that to make DNA keep stable.
In addition, the preservation liquid of combination of the present invention, chemical property is stable, and one section is preserved by preservation liquid of the present invention
After time (such as 50 days), animal tissue carries out conventional method DNA extractions, can obtain the genomic DNA of large fragment, comparatively
Formaldehyde, ethanol, which preserve, can not all realize that (time that usual sample formaldehyde preserves is slightly long, and individual is difficult to be digested again, can not
Extract DNA；Formaldehyde can make DNA and protein, covalent bond is especially produced with histone DNA releases are encountered difficulties；
Crosslinking between large biological molecule, which is exaggerated, may shear nucleic acid backbone, cause DNA fragmentation；Covalent bond is produced between DNA molecular
Hinder effectively amplification；By modification of the formaldehyde to DNA base sequence, the length of amplifiable fragment is limited, therefore, formaldehyde is protected
Deposit the extraction and amplification less practicality for DNA)；And the present invention preserves progress DNA extractions after liquid, DNA is easier to discharge
(DNA degradation be mainly endogenous dna enzyme and pollution exogenous DNA enzyme effect caused by, ethanol fix preservation make sample DNA enzyme become
Property inactivation, it is impossible to degradation of dna, extract ethanol fix preserve material DNA when, generally require before digestion with TE solution soak
Dissolve ethanol, otherwise ethanol will reduce the activity of Proteinase K, and Proteinase K activity is affected, and make the broken of cell and histone
Bad degree reduces, and directly affects DNA release).
In the preferred embodiment of the invention, DNA class loadings sample is selected from the tissue sample of animal.The implementation being more highly preferred to
In scheme, DNA class loadings sample is selected from such as Mammalia (Mammalia), Yi guiding principles (Echiurida), Crustachia (Crustacean)
With the tissue sample of the animal such as Bivalvia (Bivalvia), such as musculature.In the embodiment being more highly preferred to, such as Mammalia
(Mammalia) rabbit (Oryctolagus cuniculus), Yi guiding principles (Echiurida) Urechis uniconctus (Urechis
Unicinctus), Crustachia (Crustacean) Penaeus Vannmei (Penaeus vannamei) and Bivalvia (Bivalvia)
The musculature of Ruditapes philippinarum (Ruditapes philippinarum).
In the preferred embodiment of the invention, in DNA class loading Sample storage liquid, ammonium oxalate saturated solution, ethylenediamine tetraacetic
Acetic acid 0.05~2mM of disodium, trihydroxy 10~50mM of aminomethane, Triton X-100 0.2~0.5% (v/v), chlorine
Change sodium 0.5~1.5% (w/w), pH value is controlled between 7.2~8.1.
Inventor has found that the concentration for preserving disodium ethylene diamine tetraacetate in liquid can not be too high, although it can effectively be chelated
Mg2+To suppress the activity of DNA enzymatic, but EDTA in itself the degraded for animal tissue and it is discrete have facilitation, it is corresponding to reduce
EDTA content, the discrete of animal tissue is reduced, while ensure its effect of suppression to DNA enzymatic under the auxiliary of saturated oxalic acid solution
Fruit, it is the another improvement of the present invention.
Sodium chloride concentration of the present invention is 0.5~1.5% (w/w), and animal tissue can be effectively maintained in this concentration range
Form stable, prevent that tissue loss is discrete, while this concentration range is beneficial to maintain stable in DNA chronic tissues and preserved.
In highly preferred embodiment, in DNA class loading Sample storage liquid, 60 DEG C of ammonium oxalate saturated solutions, trihydroxies
Aminomethane 0.24% (w/v), disodium ethylene diamine tetraacetate 0.074% (w/v), sodium chloride 0.9% (w/v), polyethylene glycol are pungent
Base phenyl ether 0.3% (v/v) and deionized purified water composition.
In another embodiment of the present invention, a kind of preparation method of DNA class loadings Sample storage liquid is disclosed, including such as
Lower step：
(1) deionized water of certain volume is measured, is heated to 60 DEG C, adds 80~140g (NH4)2C2O4, by measuring
Tris、EDTA-Na2.2H2O, NaCl, Triton X-100, fully mix.
(2) solution adjusts pH value to 7.2~8.1, is settled to 1.0L.
(3) autoclaving packing is standby.
In preferred embodiment, a kind of preparation method of DNA class loadings Sample storage liquid, comprise the following steps：
(1) 900mL deionized waters are measured, are heated to 60 DEG C, sequentially add 140g (NH4)2C2O4、2.42gTris、0.74g
EDTA-Na2.2H2O, 9.0g NaCl, 3.0mL Triton X-100, are sufficiently stirred mixing.
(2) pH value is adjusted to 7.5 with NaOH solution, be settled to 1.0L.
(3) 115 DEG C of sterilizing 20min of high pressure, are dispensed standby immediately.
In another embodiment of the present invention, the application of above-mentioned DNA class loadings Sample storage liquid, the application are also disclosed
The application being included in DNA sequencing, molecule genetics research and/forensic identification.
Based on above-mentioned application, the invention discloses a kind of store method of DNA class loadings sample, including by DNA class loadings
Sample immerses the step of preserving liquid.
In preferred embodiment, preserve liquid and preserve volume ratio >=3 of tissue:1.The dosage of the present invention for preserving liquid
Considerably less than ethanol consumption, it is easy to the lightness of collecting sample to preserve；And preservation liquid stable performance of the present invention, it is fixed to protect
During depositing liquid is preserved without changing.
In preferred embodiment, DNA class loadings sample, which immerses, preserves liquid after room temperature preservation or even lower in 4 DEG C
Temperature preserves.In the embodiment of the present invention, animal tissue's sample, which immerses, to be preserved in liquid in the preservation 2 months of (20~30 DEG C) of room temperature, is moved
The disappearance of thing tissue sample is less, and can effectively extract large fragment DNA.
DNA class loadings sample collection is also disclosed in one embodiment of the present of invention and preserves kit, including above-mentioned preservation
Liquid, sterile storage containers.
One embodiment of the present of invention also discloses a kind of method of DNA molecular detection, including uses above-mentioned preservation liquid pair
The step of tissue sample is preserved.
Also include the step of DNA extractions in preferred embodiment, in the method for DNA molecular detection；In the present invention, tissue
Sample storage liquid ingredient is smaller for the influence of follow-up DNA extracts reagents, and beneficial to DNA releases in tissue, it is only necessary to
Operated after chorista according to conventional DNA extraction method.
Further, the method for DNA molecular detection also carries out the step of molecular Biological Detection using the DNA of extraction
Suddenly.
Embodiment 1
One embodiment of the invention, its component and content are as shown in table 1 below,
Table 1 preserves formula of liquid
| Component | Content |
| EDTA-Na2.2H2O | 0.74g |
| NaCl | 9.0g |
| Triton X-100 | 3.0mL |
| Tris | 2.42g |
| (NH4)2C2O4 | 140g |
| Deionized water | 1.0L |
In this formula, the main function of each composition is：
Tris is weak base, and its aqueous solution can allow the pH value for preserving liquid to maintain alkalescent, and DNA is relatively stable under the environment.
EDTA-Na2.2H2O is as Ca2+、Mg2+Chelating agent, can chelate makes DNA enzymatic play a role necessary Mg2+, from
And DNA enzymatic is set not play a role.
Triton X-100 are a kind of nonionic surface active agent, and it can dissolve the lipid on cell membrane, thin to increase
Permeability of the after birth to ion.
(NH4)2C2O4Can be with metal ion such as Mg2+Reaction, form the oxalates for being slightly soluble in water.Used in the present invention
High concentration (NH4)2C2O4The growth of microorganism can be suppressed, changed so as to avoid preserving the acid-base property in liquid caused by microorganism growth
Become and degraded of the metabolite of microorganism to DNA in tissue.
Embodiment 2
1. the preservation liquid prepared with embodiment 1 (presses animal tissue:Preserve liquid=1:10 ratio) under room temperature condition respectively
Preserve Mammalia (Mammalia) rabbit (Oryctolagus cuniculus), Yi guiding principles (Echiurida) Urechis uniconctus
(Urechis unicinctus), Crustachia (Crustacean) Penaeus Vannmei (Penaeus vannamei) and Bivalvia
(Bivalvia) musculature of Ruditapes philippinarum (Ruditapes philippinarum), while tissue is pressed into animal tissue:
Absolute ethyl alcohol=1:10 ratio preserves above-mentioned animal tissue, room temperature storage.Rabbit, Urechis uniconctus, Penaeus Vannmei and Fei Lv
Guest clam son is that commercially available purchase obtains.
2. with DNA extraction kit (EasyPure Marine Animal Genomic DNA Kit, north after preserving 50 days
Jing Quanshijin Bioisystech Co., Ltd), require extraction genomic DNA in strict accordance with specification.
3. extract product to be detected with 1% agarose electrophoresis, the μ l loadings of Marker 5, the DNA extraction μ l loadings of product 2, electricity
Result of swimming is as shown in Figure 1.
4. result shows that the DNA electrophoretic bands extracted from the tissue for preserving liquid preservation are clear, without diffusing phenomenon, and use
The tissue DNA degraded that absolute ethyl alcohol preserves is serious, without obvious main band.Illustrate using this preservation liquid energy enough well in room temperature
Under the conditions of preserve animal tissue at least 50 days, and be better than traditional ethanol preservation method, therefore the suitable room temperature condition of this preservation liquid
The long-term preservation of lower animal tissue.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (10)
1. a kind of DNA class loadings Sample storage liquid, its composition by ammonium oxalate, trihydroxy aminomethane, disodium ethylene diamine tetraacetate,
Sodium chloride, Triton X-100 and deionized purified water composition.
2. DNA class loadings Sample storage liquid according to claim 1, it is characterised in that the DNA class loadings sample is selected from
The tissue sample of animal；
Preferably, DNA class loadings sample is selected from Mammalia (Mammalia), Yi guiding principles (Echiurida), Crustachia
(Crustacean) and Bivalvia (Bivalvia) tissue sample, be more highly preferred to, tissue sample is musculature；
Preferably, DNA class loadings sample is selected from rabbit (Oryctolagus cuniculus), Urechis uniconctus (Urechis
Unicinctus), Penaeus Vannmei (Penaeus vannamei) and Ruditapes philippinarum (Ruditapes philippinarum)
Musculature.
3. DNA class loadings Sample storage liquid according to claim 1 or 2, it is characterised in that DNA class loading Sample storages
In liquid, ammonium oxalate saturated solution, 0.05~2mM of disodium ethylene diamine tetraacetate, trihydroxy 10~50mM of aminomethane, polyethylene glycol
Octyl phenyl ether 0.2~0.5% (v/v), sodium chloride 0.5~1.5% (w/w), pH value are controlled between 7.2~8.1.
4. DNA class loadings Sample storage liquid according to claim 1 or 2, it is characterised in that DNA class loading Sample storages
In liquid, ammonium oxalate saturated solution, trihydroxy aminomethane 0.24% (w/v), disodium ethylene diamine tetraacetate 0.074% (w/v), chlorine
Change sodium 0.9% (w/v), Triton X-100 0.3% (v/v).
5. the preparation method of the DNA class loading Sample storage liquid described in claim any one of 1-5, it is characterised in that including such as
Lower step：
(1) deionized water of certain volume is measured, is heated to 50 DEG C~60 DEG C, adds 80~140g (NH4)2C2O4, by measuring
Tris、EDTA-Na2.2H2O, NaCl, Triton X-100, fully mix；
(2) solution adjusts pH value to 7.2~8.1, is settled to 1.0L；
(3) autoclaving packing is standby.
6. a kind of preparation method of DNA class loadings Sample storage liquid, comprises the following steps：
(1) 900mL deionized waters are measured, are heated to 60 DEG C, sequentially add 140g (NH4)2C2O4、2.42g Tris、0.74g
EDTA-Na2.2H2O, 9.0g NaCl, 3.0mL Triton X-100, are sufficiently stirred mixing；
(2) pH value is adjusted to 7.5 with NaOH solution, be settled to 1.0L；
(3) 115 DEG C of sterilizing 20min of high pressure, are dispensed standby immediately.
7. the application of the DNA class loading Sample storage liquid described in claim any one of 1-5, the application are included in molecular genetic
Learn the application in research, DNA sequencing and/forensic identification.
8. a kind of store method of DNA class loadings sample, including DNA class loadings sample is immersed into any one of claim 1-5 institute
The step of DNA class loading Sample storage liquid stated；
Preferable volume ratio >=3 for preserving liquid and tissue sample:1；
Preferably；DNA class loadings sample, which immerses, preserves liquid after room temperature preservation or in 4 DEG C of even lower temperature preservations.
9. a kind of DNA class loadings sample collection preserves kit, including the DNA class loading samples described in claim any one of 1-5
Product preserve liquid, sterile storage containers.
10. a kind of method of DNA molecular detection, including using the DNA class loading Sample storages described in claim any one of 1-5
The step of liquid preserves to tissue sample；
Preferably, the step of also including DNA extractions in the method for DNA molecular detection；
Further preferable, the method for DNA molecular detection also carries out the step of molecular Biological Detection using the DNA of extraction
Suddenly.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111188094A (en) * | 2020-02-24 | 2020-05-22 | 南京诺唯赞生物科技有限公司 | Sequencing library construction method and kit for pathogenic microorganism detection |
| CN111718927A (en) * | 2020-06-24 | 2020-09-29 | 南京诺唯赞生物科技股份有限公司 | Preservation solution for improving stability of nucleic acid and application thereof |
| CN113218723A (en) * | 2021-03-16 | 2021-08-06 | 吴敏 | Composition for microscopic observation and preparation method and application thereof |
| CN113455495A (en) * | 2021-05-16 | 2021-10-01 | 苏州标点生物科技有限公司 | Tumor tissue DNA and RNA preservation solution |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105961375A (en) * | 2016-07-13 | 2016-09-28 | 北京中科唯新生物医学研究所有限公司 | Saliva preserving fluid, preparation method and usage thereof |
-
2017
- 2017-08-21 CN CN201710716506.6A patent/CN107447007B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105961375A (en) * | 2016-07-13 | 2016-09-28 | 北京中科唯新生物医学研究所有限公司 | Saliva preserving fluid, preparation method and usage thereof |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111188094A (en) * | 2020-02-24 | 2020-05-22 | 南京诺唯赞生物科技有限公司 | Sequencing library construction method and kit for pathogenic microorganism detection |
| CN111188094B (en) * | 2020-02-24 | 2020-10-16 | 南京诺唯赞生物科技股份有限公司 | Sequencing library construction method and kit for pathogenic microorganism detection |
| CN111718927A (en) * | 2020-06-24 | 2020-09-29 | 南京诺唯赞生物科技股份有限公司 | Preservation solution for improving stability of nucleic acid and application thereof |
| CN113218723A (en) * | 2021-03-16 | 2021-08-06 | 吴敏 | Composition for microscopic observation and preparation method and application thereof |
| CN113455495A (en) * | 2021-05-16 | 2021-10-01 | 苏州标点生物科技有限公司 | Tumor tissue DNA and RNA preservation solution |
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| Publication number | Publication date |
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| CN107447007B (en) | 2019-04-09 |
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