CN107641597A - Denaturation hybridizing box and its application method for FISH - Google Patents
Denaturation hybridizing box and its application method for FISH Download PDFInfo
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- CN107641597A CN107641597A CN201711007000.4A CN201711007000A CN107641597A CN 107641597 A CN107641597 A CN 107641597A CN 201711007000 A CN201711007000 A CN 201711007000A CN 107641597 A CN107641597 A CN 107641597A
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- 238000004925 denaturation Methods 0.000 title claims abstract description 77
- 230000036425 denaturation Effects 0.000 title claims abstract description 77
- 238000000034 method Methods 0.000 title claims abstract description 25
- 238000002474 experimental method Methods 0.000 claims abstract description 15
- 238000009396 hybridization Methods 0.000 claims description 30
- 238000010438 heat treatment Methods 0.000 claims description 12
- 238000002955 isolation Methods 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 6
- 238000007901 in situ hybridization Methods 0.000 abstract description 4
- 239000000523 sample Substances 0.000 description 25
- 210000000349 chromosome Anatomy 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 210000002700 urine Anatomy 0.000 description 2
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 239000004411 aluminium Substances 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000006059 cover glass Substances 0.000 description 1
- 230000002559 cytogenic effect Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000007769 metal material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 108700042657 p16 Genes Proteins 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 238000003793 prenatal diagnosis Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention provides a kind of denaturation hybridizing box and its application method for fluorescence in situ hybridization technique, belongs to biological technical field.The denaturation hybridizing box includes lid and box body, sets dismountable slide position-limited rack in box body, and the slide position-limited rack includes a cross spacing plate and several and vertical spacing plate that cross spacing plate is in right-angled intersection.The application method of the denaturation hybridizing box includes the placement of wet bar, slide is placed, is denatured and hybridizes.The structure of the denaturation hybridizing box is simple, easy to operate, alternative FISH instrument expensive at present, the denaturation in FISH experiments and crossover operation are conveniently and efficiently realized, while the batch detection of great amount of samples can also be realized, significantly reduces the cost of FISH detections.
Description
Technical field
It is especially a kind of for fluorescence in situ hybridization technique the present invention relates to one kind denaturation hybridizing box and its application method
Hybridizing box and its application method are denatured, belongs to biological technical field.
Background technology
FISH（Fluorescence in situ hybridization FISH）It is an emerging molecule
Cytogenetic techniques.FISH principle is：According to nucleic acid base complementary pairing principle, visited with hapten-marked DNA or RNA
Pin and the single strand nucleotide sequence complementary pairing by denaturation, go to identify that haptens is examined by the antibody with fluorophor
Survey, or probe is carried out directly to mark and combined with target sequence with fluorophor, finally directly seen using fluorescence microscope
Examine distribution situation of the target sequence in nucleus, chromosome or biopsy tissues.This technology has been widely used for moving at present
Plant Genome structural research, the analysis of variance of chromosome fine structure, viral infection assays, mankind's pre-natal diagnosis, tumorgenesis
Learn and many fields are treated in genome evolution research.
FISH technology mainly includes following steps：（1）The preparation of sample slide；（2）The pretreatment of sample slide；
（3）The denaturation and hybridization of probe and sample；（4）Slide washs；（5）As a result observe.During the entire process of FISH detections, probe
The step of denaturation and hybridization with sample are experiment most criticals, the success or not to testing result has served conclusive.
In order to complete FISH cross experiment, many laboratories generally select FISH instrument.In general original position is miscellaneous
Instrument is handed over to include several operator schemes such as denaturation/hybridization, hybridization and fixed temperature, price is 5~100,000 yuans.These hybridization
For instrument when carrying out FISH experiments, use cost is very high.And a FISH instrument maximum can detect 12 samples, work as sample
Need to configure more FISH instrument or regulation experiment step when this amount is excessive, substantially increase the negative of laboratory
Load.
The content of the invention
In view of the above-mentioned problems, it is an object of the invention to provide a kind of denaturation hybridizing box for FISH and its make
With method, FISH instrument expensive at present is substituted, conveniently and efficiently realizes the denaturation in FISH experiments and crossover operation,
The batch detection of great amount of samples can also be realized simultaneously, significantly reduce the cost of FISH detections.
To achieve the above object, the present invention uses following technical scheme：
A kind of denaturation hybridizing box for FISH, including lid and box body, box body is interior to set dismountable slide limit
Position frame, the slide position-limited rack include a cross spacing plate and several with cross spacing plate be in right-angled intersection vertical spacing
Plate.
Preferably, cross spacing plate forms detachable connection with vertical spacing plate by groove.In this way, according to slide
Width and number, can freely adjust the spacing and number of vertical spacing plate, to realize the position restriction of slide, prevent slide
Slided in box body, while different slides is isolated, avoid sample room from polluting.
Further, the beeline of the Edge Distance inboard wall of cartridge of vertical spacing plate is 0.5-1.5cm.I.e. vertical limit
The edge of position plate contacts not as cross spacing plate with inboard wall of cartridge, but has 0.5-1.5cm distance, facilitates glass
Piece picks and places.
Preferably, the surrounding in lid inner surface sets several wet bar geometrical clamps.Wet bar geometrical clamp can be by solid
Varying number and the wet bar of specification size are determined, to realize that slide is being denatured and the different humidity requirement in crossover process.To realize
Bigger humidity range, and humidity environment evenly is provided for slide, in the surrounding of tray interior, wet bar limiting card is set
Groove.
Preferably, the surrounding in lid external upper sets depression to enclose groove, the surrounding of the lower surface of box portion outside is set
Put and depression encloses the projection that groove cooperates and encloses groove.Depression encloses groove and projection and encloses groove mutual cooperation so that the change of two and the above
Property hybridizing box forms firm overlapping.It is convenient to shift multiple hybridization denaturation boxes simultaneously.
Preferably, the both sides external of box body sets handle, operating personnel are facilitated to pick and place denaturation hybridizing box.
Preferably, the center of lid external upper sets handle, facilitate opening and the closing of lid.It is more for convenience
Individual denaturation hybridizing box is overlapping, and a groove that can store handle is set on lid.
Preferably, setting temperature inductor in the bottom surface of tray interior, the temperature of box body bottom is measured, to protect
Demonstrate,prove the hybridization denaturation temperature of the slide contacted with box body bottom.
A kind of application method of denaturation hybridizing box recited above, comprises the following steps：
（1）Wet bar is placed：Experimental humidity requirement, with the wet bar limit card slot in wet the bar geometrical clamp and box body in lid
The wet bar of fixed respective numbers；
（2）Slide is placed：The slide that mounting is completed is placed on to the bottom surface of denaturation hybridizing box tray interior, and it is spacing by slide
Frame carries out position restriction and isolation to slide；
（3）Denaturation：The denaturation hybridizing box for being placed with slide is transferred in constant temperature heating device and is denatured；
（4）Hybridization：The denaturation hybridizing box for being denatured completion is transferred in constant temperature heating device and hybridized；, will after the completion of hybridization
Slide takes out from denaturation hybridizing box, that is, completes the denaturation crossover process of FISH experiment.
Preferably, step（3）Described in constant temperature heating device be water-bath, described denaturation temperature is 70-80 DEG C,
Denaturation time is 3-10 minutes.
Preferably, step（4）Described in constant temperature heating device be constant incubator, described hybridization temperature is 37-
48 DEG C, hybridization time 12-16h.
Compared with prior art, the invention has the advantages that：
（1）Provided by the present invention for the denaturation hybridizing box of FISH, set dismountable slide spacing in box body
Frame, position restriction is carried out to slide, avoids sample room from polluting.Meanwhile after denaturation and hybrid experiment is completed, slide can be limited
Position frame take out, facilitate the cleaning of tray interior, avoid because clean it is not thorough and caused by detect batch between sample pollute situation.
（2）The cross spacing plate of the present invention forms detachable connection with vertical spacing plate by groove.Due to different rule
The slide of lattice has different width, and the top edge of cross spacing plate has several grooves, can be by adjusting vertical spacing plate
Realize the uniformity with slide width in the position for inserting cross spacing plate top edge groove；In addition, for some small-scale examinations
Test, slide sample is less, can suitably reduce the number of vertical spacing plate, reduces the workload of vertical spacing plate cleaning, simultaneously
Extend its service life.
（3）In the present invention, several wet bar geometrical clamps are set in the surrounding of lid inner surface, can be by fixed different
The wet bar of quantity and specification size, to realize that slide is being denatured and the different humidity requirement in crossover process.It is bigger to realize
Humidity range, and humidity environment evenly is provided for slide, in the surrounding of tray interior, wet bar limit card slot is set.
（4）In general FISH instrument maximum can detect 12 samples.Denaturation hybridizing box provided by the invention, each
Can places 12 samples simultaneously in hybridizing box.And the present invention also sets the bumps of mutual cooperation to enclose on lid and box body
Groove, it is possible to achieve the denaturation hybridizing box of two and the above formed it is firm overlapping, it is convenient to shift multiple hybridization denaturation boxes simultaneously, you can
To realize that the FISH of more multisample is detected simultaneously, operating efficiency is drastically increased.
（5）The hybridization denaturation temperature change of FISH experiments is generally：Normal temperature → 75 DEG C → 42 DEG C.Currently used fluorescence is former
Position hybridization instrument, in order to realize the conversion of different temperatures, containing fan and heater, but stills need heating and the drop of long period
Warm process.And the denaturation hybridizing box of the new offer of this experiment, purpose temperature can be quickly transferred to according to experiment demand
Constant temperature heating device（Such as water-bath, incubator）In, the rapid temperature realized required for experiment.
（6）It is simple in construction provided by the present invention for the denaturation hybridizing box of FISH, cheap, user
Method is simple, the conventional constant temperature heating device with laboratory（Such as water-bath, incubator）It is used in combination, the fluorescence for substituting costliness is former
Position hybridization instrument, FISH denaturation and crossover operation are easily completed, significantly reduce the cost of FISH detections, and can also be same
When handle more samples, drastically increase operating efficiency.
Brief description of the drawings
Fig. 1 is the tray interior top view of denaturation hybridizing box；
Fig. 2 is the box body structure schematic diagram of denaturation hybridizing box；
Fig. 3 is the lid inner surface schematic diagram a of denaturation hybridizing box；
Fig. 4 is the lid inner surface schematic diagram b of denaturation hybridizing box；
Fig. 5 is the lid inner surface schematic diagram c of denaturation hybridizing box；
Fig. 6 is the structural representation of the lid external upper of denaturation hybridizing box；
Wherein, 1 is lid；2 be box body；3 be slide position-limited rack；11 be wet bar geometrical clamp；12 be that depression encloses groove；13 be lid handle
Hand；21 be wet bar limit card slot；22 be that projection encloses groove；23 be box body handle；31 be cross spacing plate；311 be cross spacing plate
Groove；32 be vertical spacing plate；321 be vertical spacing plate groove；Wet bar 4.
Embodiment
The present invention is described further with reference to the drawings and specific embodiments.It should be understood that these embodiments are merely to illustrate
Purpose, rather than the limitation scope of the invention.
Embodiment 1
A kind of denaturation hybridizing box for FISH, including lid and box body.As shown in figure 1, set in box body 2 removable
The slide position-limited rack 3 unloaded, slide position-limited rack 3 includes a cross spacing plate 31 and several are handed over cross spacing plate 31 in cross
The vertical spacing plate 32 of fork.The number of vertical spacing plate 32 is 4 in Fig. 1,10 slide samples can be placed, in tray interior
Bottom plate on also have slide sample position numbering, avoid slide sample from obscuring.
The box body structure of hybridizing box is denatured as shown in Fig. 2 the top edge in cross spacing plate 31 there are several cross spacings
Plate groove 311, there are a vertical spacing plate groove 321, cross spacing plate groove 311 and vertical spacing on vertical spacing plate 32
Plate groove 321 cooperates, and realizes that cross spacing plate 31 is connected with vertical spacing plate 32 in dismountable right-angled intersection formula.This
Outside, can also be by adjusting the spacing and number of vertical spacing plate, to adapt to different slide width and quantity.
In Fig. 2, the edge of vertical spacing plate 32 contacts not as cross spacing plate 31 with inboard wall of cartridge, but
There is 0.5-1.5cm distance, facilitate picking and placeing for slide.
It is slide in denaturation and crossover process as shown in Fig. 2 surrounding inside box body 2 sets wet bar limit card slot 21
The humidity environment of middle offer evenly.
In Fig. 2, the surrounding of the lower surface outside box body 2 sets projection to enclose groove 22.The both sides external of box body 2 sets box body
Handle 23, operating personnel are facilitated to pick and place denaturation hybridizing box.
The lid inner surface structure for being denatured hybridizing box as in Figure 3-5, sets several wet in the surrounding of the inner surface of lid 1
Bar geometrical clamp 11.It is different with the humidity requirement in crossover process due to being denatured in different slide samples, it can be fixed by wet bar
The fixation varying numbers of folder 11 and the wet bar 4 of specification size realize different humidity.
Wet bar geometrical clamp 11 cooperates with the wet bar limit card slot 21 in Fig. 2, can be to the humidity inside denaturation hybridizing box
Carry out larger range of regulation.
The structure of the lid external upper of hybridizing box is denatured as shown in fig. 6, the surrounding in the external upper of lid 1 is set
Depression encloses groove 12.Depression, which encloses groove 12 and encloses groove 22 with the projection in Fig. 2, to cooperate so that the denaturation of two and the above hybridizes box-like
It is overlapping into consolidating, it is convenient to shift multiple hybridization denaturation boxes simultaneously.
In Fig. 6, the center of the external upper of lid 1 sets lid handle 13, facilitates opening and the closing of lid.For side
Just multiple denaturation hybridizing boxes are overlapping, and a groove that can store handle can be set on lid（Do not shown in Fig. 6）.
In order to be measured to the temperature of box body bottom, become warm-natured with the hybridization for the slide for ensureing to contact with box body bottom
Degree, temperature inductor can be set in the bottom surface of tray interior（Do not shown in figure）.In order to monitor the humidity of tray interior, may be used also
To set humidity sensor in box body（Do not shown in figure）.
In order that slide is heated evenly in crossover process is denatured, box body inner bottom part, placement glass in denaturation hybridizing box
The region of piece needs guarantee very smooth.In order that slide can quickly achieve experiment needed for temperature, be denatured the material of hybridizing box
The metal material with high thermal conductivity, such as aluminium may be selected.
Embodiment 2
A kind of application of the denaturation hybridizing box in fluorescence in situ hybridization technique described in embodiment 1, come off with detecting in urine carefully
In the number of No. 3, No. 7, No. 17 chromosomes of born of the same parents and No. 9 chromosomes exemplified by the number of p16 gene locis.
Cast-off cells through urine capture, after the pretreatment such as hypotonic medium and pepsin, you can miscellaneous for fluorescent in situ
Hand over（FISH）Analysis.Crossover operation flow includes：
Before hybridization starts, the denaturation hybridizing box described in embodiment 1 is got out, and according to the experiment demand of reality, be denatured
The lid inner surface of hybridizing box（Fixed with wet bar geometrical clamp）It is put into a number of wet bar.In order to obtain bigger humidity, also
In the surrounding of tray interior a number of wet bar can be fixed with wet bar limit card slot.
（1）Probe prepares：（Hybridization buffer 8ul+ probes 2ul）Of short duration centrifugation after mixing.
（2）The preparation of probe and sample：10 μ l probe mixtures are dripped in slide hybridising region, are capped cover glass immediately,
With mounting adhesive edge；
（3）Slide is placed：The slide that mounting is completed, the bottom surface of denaturation hybridizing box tray interior is placed in, is limited by slide
Position frame carries out slide locations restriction, and different slide samples is isolated, and covers denaturation hybridizing box lid；
In order that slide preferably can be bonded with the bottom surface of tray interior, can be before slide placement, at the bottom of tray interior
Sterilized water is dripped in the position to be placed of face, slide, then places slide again；
（4）The denaturation of probe and sample：Denaturation hybridizing box is placed in 75 DEG C（The hybridization temperature of different samples is different, generally 70-
80℃）The common constant temperature heating device in laboratory（Such as water-bath）In 5 minutes（The hybridization time of different samples is different, typically
For 3-10 minutes）；It can place multiple denaturation hybridizing boxes according to the size and number of water-bath and be denatured simultaneously；
（5）The hybridization of probe and sample：After the completion of denaturation, by one or more denaturation hybridizing boxes it is overlapping after be transferred to 42 DEG C（No
It is different with the hybridization temperature of sample, generally 37-48 DEG C）The common constant temperature heating device in laboratory（As water-bath, constant temperature are trained
Support case etc.）Middle hybridized overnight（Generally 12-16h）；
（6）Hybridization takes out slide from hybridizing box after terminating, and carries out follow-up elution step, observes results of hybridization.
Fluorescence microscope result is shown：Results of hybridization is good, it is observed that the fluorescence signal marked on probe.
It should be noted that the preferable embodiment of the present invention is given in the specification and its accompanying drawing of the present invention, but
It is that the present invention can be realized by many different forms, however it is not limited to the embodiment described by this specification, these realities
Mode is applied not as the extra limitation to present invention, there is provided the purpose of these embodiments is made in disclosure of the invention
The understanding of appearance more thorough and comprehensive.But after the above of the present invention has been read, those skilled in the art can be to this hair
Bright to make various changes or modifications, these equivalent form of values equally fall within the application appended claims limited range.
Claims (9)
1. a kind of denaturation hybridizing box for FISH, including lid and box body, it is characterised in that set in the box body
Dismountable slide position-limited rack is put, it is in ten that the slide position-limited rack, which includes a cross spacing plate and several and cross spacing plate,
The vertical spacing plate that word intersects, the cross spacing plate form detachable connection with vertical spacing plate by groove；The box
The surrounding of interior surface sets several wet bar geometrical clamps.
2. denaturation hybridizing box according to claim 1, it is characterised in that in the Edge Distance box body of the vertical spacing plate
The beeline of wall is 0.5-1.5cm.
3. denaturation hybridizing box according to claim 1 or 2, it is characterised in that the surrounding of the tray interior sets wet bar
Limit card slot.
4. according to the denaturation hybridizing box described in claim any one of 1-3, it is characterised in that the four of the lid external upper
Depression is set to enclose groove in week, the surrounding of the box portion outside lower surface sets the projection for enclosing groove mutual cooperation with depression to enclose groove；It is described
The both sides external of box body sets handle.
5. according to the denaturation hybridizing box described in claim any one of 1-4, it is characterised in that in the lid external upper
Centre sets handle.
6. according to the denaturation hybridizing box described in claim any one of 1-5, it is characterised in that the bottom surface of the tray interior is set
Temperature inductor.
7. the application method of the denaturation hybridizing box described in a kind of any one of claim 1-6, it is characterised in that including following step
Suddenly：
（1）Wet bar is placed：Experimental humidity requirement, the wet bar of respective numbers is fixed with the wet bar geometrical clamp in lid；
（2）Slide is placed：The slide that mounting is completed is placed on to the bottom surface of denaturation hybridizing box tray interior, and it is spacing by slide
Frame carries out position restriction and isolation to slide；
（3）Denaturation：The denaturation hybridizing box for being placed with slide is transferred in constant temperature heating device and is denatured；
（4）Hybridization：The denaturation hybridizing box for being denatured completion is transferred in constant temperature heating device and hybridized；, will after the completion of hybridization
Slide takes out from denaturation hybridizing box, that is, completes the denaturation crossover process of FISH experiment.
8. the application method of denaturation hybridizing box according to claim 7, it is characterised in that step（3）Described in constant temperature
Heater is water-bath, and described denaturation temperature is 70-80 DEG C, and denaturation time is 3-10 minutes.
9. the application method of the denaturation hybridizing box according to claim any one of 7-8, it is characterised in that step（4）Middle institute
The constant temperature heating device stated is constant incubator, and described hybridization temperature is 37-48 DEG C, hybridization time 12-16h.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711007000.4A CN107641597A (en) | 2017-10-25 | 2017-10-25 | Denaturation hybridizing box and its application method for FISH |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711007000.4A CN107641597A (en) | 2017-10-25 | 2017-10-25 | Denaturation hybridizing box and its application method for FISH |
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