CN110257483A - Hybridization solution, preparation method and detection kit in situ hybridization - Google Patents
Hybridization solution, preparation method and detection kit in situ hybridization Download PDFInfo
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- 238000007901 in situ hybridization Methods 0.000 title claims abstract description 69
- 238000001514 detection method Methods 0.000 title claims abstract description 18
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 239000000243 solution Substances 0.000 claims abstract description 89
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- 239000007853 buffer solution Substances 0.000 claims abstract description 12
- LEAHFJQFYSDGGP-UHFFFAOYSA-K trisodium;dihydrogen phosphate;hydrogen phosphate Chemical compound [Na+].[Na+].[Na+].OP(O)([O-])=O.OP([O-])([O-])=O LEAHFJQFYSDGGP-UHFFFAOYSA-K 0.000 claims abstract description 12
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- 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
- C12Q1/6813—Hybridisation assays
- C12Q1/6841—In situ hybridisation
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Abstract
The present invention relates to a kind of hybridization solutions, preparation method and hybridization in situ detection kit in situ hybridization.This is used for the hybridization solution of in situ hybridization, the component including following final concentration: formamide of the concentration expressed in percentage by volume no more than 2%, the fluoride that bulking value percentage concentration is 0.1-10%, 2-4 times of concentration SSC, 1-10mM EDTA, 1-5 times of concentration Denhardts solution, the dextran sulfate that bulking value percentage concentration is 5-10%, the salmon sperm dna of 0.1-1mg/mL and 50-100mM and pH be 7.0 disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution or Tris-HCl buffer.Hybridization solution in situ hybridization of the invention can improve hybridization efficiency in the case where guaranteeing hybridization signal, shorten hybridization time.
Description
Technical field
The present invention relates to hybridization in situ technique, in particular to it is a kind of for the hybridization solution of in situ hybridization, preparation method and
Hybridization in situ detection kit.
Background technique
Gall and Pardue utilizes the probe of isotope labelling to measure nucleic acid sequence in intact cell for the first time within 1969, former
Position hybridization technique (In situ hybridization, ISH) is rapidly developed.The appearance of Cell immunohistochemical staining method,
The development of isotope-labeled nucleic acid probe is promoted again, so that fixing to conventional formalin, in the tissue of paraffin embedding
The detection of DNA and RNA be possibly realized.In the tissue fixed by formalin, formaldehyde infiltration in nucleic acid and protein it
Between, the crosslinking of DNA-DNA, DNA- protein can be caused, save antigen in original position.In situ hybridization is that molecule is hybridized and organized
It learns the technology combined and under appropriate conditions, passes through hydrogen bond knot by using label probes such as biotin, digoxin
It closes, forms DNA-DNA, DNA-RNA or RNA-RNA heteroduplex, further hybridization probe is carried out using signal amplifying system
Amplification, Color Appearance System colour developing, finally observes hybridization signal under the microscope.Signal amplifying system and Color Appearance System and immuning tissue
Chemo-antigen antibody response is identical.In situ hybridization includes colour developing in situ hybridization (Chromogenic in situ
Hybridization, CISH) and fluorescence in situ hybridization (Fluorescence in situ hybridization, FISH).It is aobvious
Color in situ hybridization application non-fluorescent label substance markers probe, such as digoxin, biotin, it is therefore desirable to which correlation-detection system will be believed
It number amplifies, if the hybridization signal after being developed the color with DAB being capable of persistence.Fluorescence in situ hybridization fluorescent label
Probe, undesired signal amplification process have the advantages that high sensitivity, can be used simultaneously multiple probes.Hybridization in situ technique can
The information of in situ detection nucleic acid level in cell, as infectious diseases, cytogenetics disease and tumour relative chromosome are different
Often, it can be above complementary to one another with immunohistochemistry in application, it has also become the conventional detection project of many hospital pathology departments.
The colour developing entire experimental implementation process of in situ hybridization includes: dewaxing, aquation, endogenous enzyme is blocked, cleaned, protease disappears
Change, clean, probe hybridization, cleaning, primary antibody incubation, cleaning, secondary antibody incubation, cleaning, colour developing, cleaning, lining contaminates, cleaning, dehydration, seals
Piece and etc..The entire experimental implementation process of fluorescence in situ hybridization includes: multiple dewaxing, aquation, hot repair, cleaning, protease digestion, clear
It washes, be dehydrated, probe hybridizes, cleans, being dehydrated, redyes.Probe hybrid process is divided into denaturation and annealing.Denaturation refers to double spiral shells
When rotation DNA molecular is heated to 92 DEG C (90 DEG C -98 DEG C) under alkaline condition or adds denaturant, the hydrogen chain of complementary base between double-strand
It unlocks and becomes single-stranded process.Annealing refers to the hydrogen bond in certain ion concentration and when gradually cooling down, between complementary base again
Connect into the process of double-stranded nucleic acid molecule (probe).In annealing process, the single stranded DNA or RNA piece of external source and sequence complementation is added
Section, can also connect to form heteroduplex with the single-stranded complementary unlocked originally, which is known as nucleic acid hybridization.It visits
Needle hybridization effect depend on signal strength and hybrid context, this again with protease digestion, hybridization temperature, probe sequence and dilution
The factors such as the hybridization solution of probe are related.
The probe hybridization duration of in situ hybridization product on the market is largely 4 to 16 hours at present, but hybridization time
Shorter, hybridization is more insufficient, and hybridization signal intensities are weaker and non-specific background is high, in this case, quick hybridization 4
Hour will likely can reduce the sensitivity of in situ hybridization, influence Clinical differential diagnosis.Therefore, how to guarantee hybridization signal intensities
In the case where, hybridization efficiency is improved, shortens hybridization time, improves the working efficiency of pathology department, it is particularly critical.
Summary of the invention
The technical problems to be solved by the present invention are: providing a kind of hybridization solution in situ hybridization, can guarantee to hybridize
In the case where signal, hybridization efficiency is improved, shortens hybridization time；
Further, the present invention also provides the preparation methods of the above-mentioned hybridization solution in situ hybridization；And one kind is based on
The hybridization in situ detection kit of the above-mentioned hybridization solution in situ hybridization.
In order to solve the above-mentioned technical problem, the technical solution adopted by the present invention are as follows:
A kind of hybridization solution in situ hybridization, the hybridization solution include the component of following final concentration: concentration expressed in percentage by volume
Formamide, bulking value percentage concentration no more than 2% are SSC, 1-10mM of the fluoride of 0.1-10%, 2-4 times of concentration
The Denhardts solution of EDTA, 1-5 times of concentration, dextran sulfate, the 0.1-1mg/mL that bulking value percentage concentration is 5-10%
Salmon sperm dna and 50-100mM and pH be 7.0 disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution or Tris-HCl buffering
Liquid.
A kind of hybridization in situ detection kit, including in situ hybridization detection probe, water and above-mentioned in situ hybridization
Hybridization solution.
A kind of preparation method of the above-mentioned hybridization solution in situ hybridization, comprising the following steps:
(1) by disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution of the sodium fluoride of 1.25g, 2.5mL and 1M, 5mL and 20 times it is dense
The sulfuric acid Portugal of the SSC solution of degree, the EDTA solution of 0.1mL and 0.5M, the Denhardts solution of 1mL and 50 times of concentration and 2.5g
Glycan mixing, the pH of the disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution are 7.0；
(2) mixed liquor for obtaining step (1) carries out centrifugal treating；
(3) salmon sperm dna of final concentration of 0.1mg/mL is added, ultrapure water is then added and carries out being settled to 50mL.
The beneficial effects of the present invention are:
(1) for the present invention in the design of hybridization solution component, using fluoride (can be from now to substitute or reduce the concentration of formamide
There is 20% to be reduced to 2% hereinafter, even 0), because formamide is noxious material, which can be reduced even without using formyl
Amine has the advantages that more environmentally-friendly；
(2) 10~26 DEG C of denaturation temperature can be reduced, is denaturalized at relatively low temperatures, more preferable holding group is conducive to
It knits form and improves the stability of DNA, reduce the destruction to DNA；Simultaneously as the concentration of probe is typically much higher than target
The concentration of DNA improves 10~26 DEG C of hybridization temperature, does not still influence probe and formed with nucleic acid in the presence of fluorine ion
Stable double-stranded chain；When hybridization solution is applied to hybridization technique as a result, hybridization temperature can be improved, and then hybridization speed can be accelerated
Degree can also improve the specificity of hybridization, reduce background, in situ hybridization is more quickly carried out, signal and background ratio are more
It is clear to add；
(3) hybridization solution of the invention can be adapted for include the different probes such as DNA and RNA hybridization, use the hybridization solution
Formula can effectively shorten hybridization time, optimize crossbreeding effect, reduce background.
Detailed description of the invention
Fig. 1 is the Kappa under the conditions of the hybridization solution in situ hybridization of the embodiment of the present invention hybridizes 90 minutes in 50 DEG C
In situ hybridization result figure；
Fig. 2 is that Kappa of the hybridization solution in situ hybridization of the embodiment of the present invention under the conditions of 37 DEG C of hybridized overnights is former
Position results of hybridization figure；
Fig. 3 is the Lambda under the conditions of the hybridization solution in situ hybridization of the embodiment of the present invention hybridizes 90 minutes in 50 DEG C
In situ hybridization result figure；
Fig. 4 is that Lambda of the hybridization solution in situ hybridization of the embodiment of the present invention under the conditions of 37 DEG C of hybridized overnights is former
Position results of hybridization figure；
Fig. 5 is that the HER2 under the conditions of the hybridization solution in situ hybridization of the embodiment of the present invention hybridizes 2 hours in 66 DEG C is glimmering
Light in situ hybridization result figure；
Fig. 6 is HER2 fluorescence of the hybridization solution in situ hybridization of the embodiment of the present invention under the conditions of 50 DEG C of hybridized overnights
In situ hybridization result figure.
Specific embodiment
To explain the technical content, the achieved purpose and the effect of the present invention in detail, below in conjunction with embodiment and cooperate attached
Figure is explained.
The most critical design of the present invention is: increasing this component of fluoride in hybridization solution component to reduce or replace first
Amide component.
Fig. 1-6 is please referred to, a kind of hybridization solution in situ hybridization, the hybridization solution includes the component of following final concentration:
Formamide of the concentration expressed in percentage by volume no more than 2%, bulking value percentage concentration are the fluoride of 0.1-10%, 2-4 times of concentration
The Denhardts solution of EDTA, 1-5 times of concentration of SSC, 1-10mM, the sulfuric acid Portugal that bulking value percentage concentration is 5-10% are poly-
Disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution that sugar, the salmon sperm dna of 0.1-1mg/mL and 50-100mM and pH are 7.0 or
Tris-HCl buffer.
A kind of hybridization in situ detection kit, including in situ hybridization detection probe, water and above-mentioned in situ hybridization
Hybridization solution.
A kind of preparation method of the above-mentioned hybridization solution in situ hybridization, comprising the following steps:
(1) by disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution of the sodium fluoride of 1.25g, 2.5mL and 1M, 5mL and 20 times it is dense
The sulfuric acid Portugal of the SSC solution of degree, the EDTA solution of 0.1mL and 0.5M, the Denhardts solution of 1mL and 50 times of concentration and 2.5g
Glycan mixing, the pH of the disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution are 7.0；
(2) mixed liquor for obtaining step (1) carries out centrifugal treating；
(3) salmon sperm dna of final concentration of 0.1mg/mL is added, ultrapure water is then added and carries out being settled to 50mL.
Now the main function mechanism of the hybridization solution in situ hybridization of the invention is illustrated:
The hybridization solution and conventional hybridization liquid phase ratio of in situ hybridization of the present invention, increase this component of fluoride to reduce or take
For formamide component.
As the atoms such as fluorine atom and oxygen, nitrogen, chlorine, stable hydrogen bond can be formed with hydrogen atom, hydrogen bond is to maintain DNA double
The primary factor of helical structure stability.Wherein the atomic radius of fluorine and hydrogen atom size are closest, are capable of forming than other
The stronger hydrogen bonds of atoms such as what atom (oxygen, nitrogen, chlorine, sulphur, phosphorus).Fluorine ion is added in hybridization solution, can effectively prevent opening
DNA double stock chain, restore the bifilar chain structure of script, the DNA after making denaturation is maintained at the state of bifilar chain separation, and probe is given to be situated between
Enter hybridization and higher probability is provided.Fluorine ion also promotes the dissociation of DNA double stock chain simultaneously, so that DNA during in situ hybridization
Denaturation temperature can carry out at a lower temperature, be conducive to the stability of DNA and probe, while keep tissue morphology.
As can be seen from the above description, the beneficial effects of the present invention are:
(1) for the present invention in the design of hybridization solution component, using fluoride (can be from now to substitute or reduce the concentration of formamide
There is 20% to be reduced to 2% hereinafter, even 0), because formamide is noxious material, which can be reduced even without using formyl
Amine has the advantages that more environmentally-friendly；
(2) 10~26 DEG C of denaturation temperature can be reduced, is denaturalized at relatively low temperatures, more preferable holding group is conducive to
It knits form and improves the stability of DNA, reduce the destruction to DNA；Simultaneously as the concentration of probe is typically much higher than target
The concentration of DNA improves 10~26 DEG C of hybridization temperature, does not still influence probe and formed with nucleic acid in the presence of fluorine ion
Stable double-stranded chain；When hybridization solution is applied to hybridization technique as a result, hybridization temperature can be improved, and then hybridization speed can be accelerated
Degree can also improve the specificity of hybridization, reduce background, in situ hybridization is more quickly carried out, signal and background ratio are more
It is clear to add；
(3) hybridization solution of the invention can be adapted for include the different probes such as DNA and RNA hybridization, use the hybridization solution
Formula can effectively shorten hybridization time, optimize crossbreeding effect, reduce background.
Hybridization solution of the invention is suitable for different in situ hybridization probes, such as DNA in situ hybridization probe, RNA in situ hybridization
The preparation of probe and fluorescence in situ hybridization probe.
The present invention is described in further details combined with specific embodiments below.
Embodiment one: Kappa and Lambda hybridization in situ detection kit
1, hybridization solution prepares (50mL):
(1) it weighs 1.25g sodium fluoride and 50mL centrifuge tube is added；
(2) take 1M disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution pH7.0 solution of 2.5mL that 50mL centrifuge tube is added；
(3) take 5mL 20x SSC solution that 50mL centrifuge tube is added；
(4) take the 0.5M EDTA solution of 0.1mL that 50mL centrifuge tube is added；
(5) take the 50x Denhardts solution of 1mL that 50mL centrifuge tube is added；
(6) it weighs 2.5g dextran sulfate addition 50mL centrifuge tube and mixing fullys shake, 1000rpm is centrifuged 5 minutes；
(7) final concentration of 0.1mg/mL salmon sperm dna is added；
(8) plus ultrapure water constant volume is in 50mL.
Finally, with the hybridization solution respectively to Kappa and Lambda probe dilution at final concentration 1ng/uL.
2, experimental method
2.1 slicing treatments: histotomy is toasted in 60-65 DEG C 60 minutes and above (suggest experiment every time while adding positive
Property, negative control piece).
2.2 dewaxings and aquation: histotomy is placed in dewaxing liquid and is impregnated 5 minutes, is repeated 3 times；100% alcohol impregnates 2 points
Clock is repeated 1 times；PBS cleaning solution impregnates 2 minutes.
Appropriate blocking agent working solution is added dropwise in 2.3 every slices, is advisable with that can cover the amount entirely organized, is incubated at room temperature 5 points
Clock.Pure water rinsing 30 seconds.
2.4 digestion enzymic digestions: drying is sliced, then the liquid of tissue block edge is blotted with blotting paper, with immunohistochemistry pen edge
Mark the region that blocks water in histotomy edge.Slice is placed in wet box, and digestive ferment (about 50 μ L, according to the increase and decrease of tissue size) are added dropwise,
It is placed in 37 DEG C of insulating boxs and is incubated for 5-15 minutes (it is recommended that 10 minutes, the specific time is related with organization type and slice thickness).PBS is clear
Washing lotion is impregnated 2 minutes.
Appropriate Kappa or Lambda chain probe (about 10 μ L, according to the increase and decrease of tissue size) are added dropwise in 2.5 every slices, and add
Coverslip (before probe is added dropwise, slice can use 100% dehydration of alcohol, be air-dried).
2.6 slices are put into wet box, and wet box is put into 90 minutes in 50 DEG C of insulating boxs and is denaturalized and is hybridized；Or 37 DEG C
Denaturation and hybridized overnight.
2.7PBS soaking at room temperature slice, carefully removes coverslip, continues immersion 4 × 3 minutes.
2.8 get rid of cleaning solution, and appropriate primary antibody (about 50 μ L, according to the increase and decrease of tissue size) are added dropwise, are incubated at room temperature 30 minutes,
PBS impregnates 3 × 3 minutes.
2.9 get rid of cleaning solution, and immune colour reagent-A liquid in right amount is added dropwise, and (blocking agent, about 50 μ L increase according to tissue size
Subtract), it is incubated at room temperature 20 minutes, PBS impregnates 3 × 3 minutes.
2.10 get rid of cleaning solution, and immune colour reagent-B liquid in right amount is added dropwise, and (polymer, about 50 μ L increase according to tissue size
Subtract), it is incubated at room temperature 20 minutes, PBS impregnates 3 × 3 minutes.
2.11 get rid of cleaning solution, and appropriate DAB developing solution is added dropwise and is allowed to that sample is completely covered, and dyeing at room temperature 5 minutes (can be
Microscopically observation develops the color situation with appropriate adjustment dyeing time).
2.12 redye: pure water rinsing 30 seconds, drying was added dropwise appropriate haematoxylin dyeing liquid and is allowed to that sample, room temperature is completely covered
Lower dyeing 3 minutes.
2.13 are washed with differentiation liquid (hydrochloride alcohol solution) and are removed within 20-30 seconds extra haematoxylin, and pure water rinsing 30 seconds, PBS leaching
Bubble returns indigo plant in 3 minutes.
2.14 dehydrations, transparent, mounting: slice successively graded ethanol (concentration from low to high 75%, 95%, 100%,
100%) it is respectively impregnated in 2 minutes；Mounting is carried out using fragrance mountant, the slice after mounting is placed in sheet of drying in the air.
3, application example one
Application example one is shown in the fluoride ion hybridization solution, is free of formamide, be joined 2.5% sodium fluoride.With
The hybridization solution prepare Kappa probe be used in situ hybridization when, hybridization with denaturation use a temperature, 50 DEG C of quick hybridization
Lower 90 minutes signal strengths are shown in Figure 1, and the signal strength of 37 DEG C of overnight hybridizations is shown in Figure 2.With the hybridization solution
When the Lambda probe of preparation is used in situ hybridization, hybridization uses a temperature with denaturation, 90 points at 50 DEG C of quick hybridization
The signal strength of clock is shown in Figure 3, and the signal strength of 37 DEG C of overnight hybridizations is shown in Figure 4.According to Fig. 1-4 it is found that with
When Kappa the and Lambda probe that the hybridization solution is prepared is used in situ hybridization, hybridization uses a temperature with denaturation, quickly miscellaneous
Hand over the signal strength that can reach 37 DEG C of overnight hybridizations at 50 DEG C for 90 minutes.Therefore, hybridization solution of the invention is advantageous in that
It reduces denaturation temperature simultaneously and improves hybridization temperature, answered so that whole process only needs a temperature that can be rapidly achieved
Some sensitivity.
Two: HER2 fluorescence in situ hybridization of embodiment
1, hybridization solution prepares (50mL):
(1) it weighs 1.25g sodium fluoride and 50mL centrifuge tube is added；
(2) take 1M disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution pH7.0 solution of 2.5mL that 50mL centrifuge tube is added；
(3) take 5mL 20x SSC solution that 50mL centrifuge tube is added；
(4) take the 0.5M EDTA solution of 0.1mL that 50mL centrifuge tube is added；
(5) take the 50x Denhardts solution of 1mL that 50mL centrifuge tube is added；
(6) it weighs 5g dextran sulfate addition 50mL centrifuge tube and mixing fullys shake, 1000rpm is centrifuged 5 minutes；
(7) final concentration of 0.2mg/mL salmon sperm dna is added；
(8) plus ultrapure water constant volume is in 50mL.
Finally, preparing HER2 probe, including final concentration 10ng/uL HER2 green fluorescence probe, final concentration with hybridization solution
1.5ng/uL CEN17 fluorescent orange probe, final concentration 1mg/mL mankind Cot-1DNA.
2, experimental method
Slide is put into immersion 3 × 10 minutes in dimethylbenzene (or dewaxing liquid) by 2.1.
Slide is put into 100%, 100%, 95% by 2.2, and 75% alcohol respectively impregnates 5 minutes, and pure water 2 × 2 minutes.
2.3 preheat citric acid in advance repairs liquid to 80 ± 2 DEG C, and slide is put into citric acid reparation liquid and is incubated for 30 minutes.
2.4 are immersed in slide in pure water 2 × 2 minutes, and slide is taken out from pure water, wipe moisture extra on slide.
2.5 slices are placed in wet box, and digestion enzyme solutions are added dropwise, and are placed in 37 DEG C of insulating boxs and are incubated for 15 minutes.Cleaning solution (2 ×
SSC it) impregnates 5 minutes.Deionized water is rinsed 1 minute.
2.6 dehydrations: 75%, 95%, it is sequentially placed 1 minute in 100% ethanol solution, is air-dried slice.
2.7 take out HER2 probe from refrigerator, are put in room temperature (18-25 DEG C), are protected from light.Before uncapping, brief centrifugation.Use liquid relief
Device inhales 10 μ L probes to each sample.Ensure not have on slide covered after bubble.Coverslip is sealed with rubber adhesive.
2.8 are put into slide in hybridization instrument, are denaturalized at 66 DEG C and are denaturalized and hybridize at hybridizing 2~3 hours or 50 DEG C
Overnight.
2.9 careful removing rubber weather strips.Slide is put into the washing buffer that 37 DEG C preheat, and (2 × SSC contains 0.1%
NP-40), impregnate 1-3 minute, carefully remove coverslip.
2.10 again wash the washing buffer that slide is preheated with 37 DEG C 2 × 5 minutes.
Slide is sequentially placed into 75%, 95% by 2.11, each 1 minute in 100% ethanol solution, is protected from light and is dried sample.
2.12 25 μ L DAPI of drop are redyed on liquid to biopsy tissues, and covered avoids bubble, and dark place is incubated for 15 minutes.
Fluorescence microscopy is under the microscope.
3, application example two
Application example two is shown in the hybridization solution of the fluoride ion, is free of formamide, be joined 2.5% sodium fluoride.With
When the HER2 fluorescence in situ hybridization probe that the hybridization solution is prepared is used in situ hybridization, hybridization uses a temperature with denaturation,
The signal strength of 2 hours is shown in Figure 5 at 66 DEG C of quick hybridization, and the signal strength of 50 DEG C of hybridized overnights is referring to Fig. 6 institute
Show.According to Fig. 5-6 it is found that when being used in situ hybridization with the HER2 fluorescence in situ hybridization probe that the hybridization solution is prepared, hybridization and change
Property use a temperature, the signal strength of hybridized overnight is all very clear bright at 2 hours of quick hybridization and 50 DEG C at 66 DEG C
It is aobvious, without non-specific background.Therefore, hybridization solution of the invention, which is advantageous in that, reduces denaturation temperature simultaneously and improves
Hybridization temperature, so that whole process only needs a temperature that can be rapidly achieved due sensitivity.
In conclusion provided by the present invention for the hybridization solution DNA in situ hybridization probe of in situ hybridization, RNA in situ hybridization
The preparation of probe and fluorescence in situ hybridization probe can improve hybridization temperature reducing denaturation temperature simultaneously, so that whole
A process only needs a temperature that can be rapidly achieved due sensitivity.
The above description is only an embodiment of the present invention, is not intended to limit the scope of the invention, all to utilize this hair
Equivalents made by bright specification and accompanying drawing content are applied directly or indirectly in relevant technical field, similarly include
In scope of patent protection of the invention.
Claims (6)
1. a kind of hybridization solution in situ hybridization, which is characterized in that the hybridization solution includes the component of following final concentration: volume
Formamide of the percentage concentration no more than 2%, bulking value percentage concentration be the fluoride of 0.1-10%, 2-4 times of concentration SSC,
The Denhardts solution of EDTA, 1-5 times of concentration of 1-10mM, the dextran sulfate that bulking value percentage concentration is 5-10%,
The salmon sperm dna and 50-100mM and pH of 0.1-1mg/mL be 7.0 disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution or
Tris-HCl buffer.
2. the hybridization solution according to claim 1 in situ hybridization, which is characterized in that the fluoride be sodium fluoride,
At least one of potassium fluoride and ammonium fluoride.
3. a kind of hybridization in situ detection kit, which is characterized in that including in situ hybridization detection probe, water and claim 1-2
The hybridization solution of in situ hybridization is used for described in any one.
4. hybridization in situ detection kit according to claim 3, which is characterized in that the in situ hybridization detection probe
Final concentration of 1ng/uL.
5. the preparation method described in a kind of claim 1-2 any one for the hybridization solution of in situ hybridization, which is characterized in that
The following steps are included:
(1) by disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution of the sodium fluoride of 1.25g, 2.5mL and 1M, 5mL and 20 times of concentration
The dextran sulfate of the EDTA solution of SSC solution, 0.1mL and 0.5M, the Denhardts solution of 1mL and 50 times of concentration and 2.5g
Mixing, the pH of the disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution are 7.0；
(2) mixed liquor for obtaining step (1) carries out centrifugal treating；
(3) salmon sperm dna of final concentration of 0.1mg/mL is added, ultrapure water is then added and carries out constant volume.
6. the preparation method of the hybridization solution according to claim 5 in situ hybridization, which is characterized in that the step
(2) centrifugal treating in specifically: be centrifuged 5 minutes under the conditions of 1000rpm.
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| CN112626180A (en) * | 2020-12-30 | 2021-04-09 | 北京思尔成生物技术有限公司 | DNA hybridization enhancing solution and preparation method and application thereof |
| WO2023206356A1 (en) * | 2022-04-29 | 2023-11-02 | 京东方科技集团股份有限公司 | Reagent and detection method for fluorescence in situ hybridization |
| CN117051114A (en) * | 2023-10-13 | 2023-11-14 | 卡秋(江苏)生物科技有限公司 | In situ hybridization probe set for Kappa and Lambda light chain mRNA detection |
| CN117887813A (en) * | 2023-11-28 | 2024-04-16 | 首都医科大学附属北京口腔医院 | Hybridization buffer solution and application |
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| CN112626180A (en) * | 2020-12-30 | 2021-04-09 | 北京思尔成生物技术有限公司 | DNA hybridization enhancing solution and preparation method and application thereof |
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| CN117887813A (en) * | 2023-11-28 | 2024-04-16 | 首都医科大学附属北京口腔医院 | Hybridization buffer solution and application |
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