CN105603063B - A method of display spotted maigre chromosome morphology feature - Google Patents
A method of display spotted maigre chromosome morphology feature Download PDFInfo
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- CN105603063B CN105603063B CN201510929377.XA CN201510929377A CN105603063B CN 105603063 B CN105603063 B CN 105603063B CN 201510929377 A CN201510929377 A CN 201510929377A CN 105603063 B CN105603063 B CN 105603063B
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Abstract
The invention discloses a kind of methods for showing spotted maigre chromosome morphology feature, choose spotted maigre, are injected using Three-ball method, hypotonic with KCl solution, then are fixed with fixer, then drip the aging on clean slide, and the slide of aging is made；Spotted maigre fin ray DNA is extracted with cell/tissue genome DNA extracting reagent kit；Spotted maigre fin ray DNA, FISH probe obtained are marked with nick translation method；The slide of aging is denaturalized in formamide, then the slide being denaturalized is made with ethanol dehydration；FISH probe is denaturalized；The FISH probe of denaturation is added on the slide being denaturalized, is hybridized in insulating box；Again through amplification, washing, signal detection.The present invention, which provides a kind of short-cut method, can make spotted maigre chromosome that special fluorescence distribution feature be presented, and improve the efficiency and accuracy of the identification of spotted maigre chromosome, solve the problems, such as that spotted maigre chromosome identification mark is poor.
Description
Technical field
The present invention relates to chromosome analysis technical field, the side of specifically a kind of display spotted maigre chromosome morphology feature
Method.
Background technique
Chromosome is the carrier of inhereditary material.Chromosome analysis is the important content of genetics research.The resolution of chromosome
It is the basis for studying chromosome with identification.
Traditional dyeing body identification rely primarily on two parameters of arm ratio and chromosome relative length differentiate homologue with
Nonhomologous chromosome.Grow up in higher vertebrate G band technology can make chromosome prolong the longitudinal axis show it is light and dark
Striped, wherein homologue streak feature is similar, and the streak feature of nonhomologous chromosome is different.Therefore, the display of G band mentions
The high efficiency and accuracy of the identification and pairing of chromosome.However, since Fish genomes structure and higher vertebrate are deposited
In difference, successfully show that the example of G- band is seldom at present.In spotted maigre, there is not yet successfully showing G band or other multiple bands
Report.Based on the FISH of chromosome specific probe, it can make homologue that similar fluorescence distribution feature be presented, and it is non-homogeneous
Different fluorescence distribution features is presented in chromosome, equally can be used for the identification of assisted staining body and pairing.However, due to separation
Chromosome specific probe is at high cost, and the R&D cycle is long, and fish are there is not yet relevant report at present.Currently, for recognizing spotted maigre dye
Color can only rely on a small number of morphological features such as arm ratio, relative length, nucleolus organizer region, position, domain.
Summary of the invention
The purpose of the present invention is to provide the display spotted maigre dyes of a kind of efficiency of spotted maigre chromosome identification and accuracy
The method of colour solid morphological feature, to solve the problems mentioned in the above background technology.
To achieve the above object, the invention provides the following technical scheme:
A method of display spotted maigre chromosome morphology feature, steps are as follows:
One, prepared by chromosome
Spotted maigre is chosen, is injected using Three-ball method；Preceding two needle focuses on pectoral fin base portion injection 0.6-1.0ml party by every 100g fish
Join the mixed liquor for decocting juice and BSA, injects the second needle after injecting the first needle 15h；Third needle be before sampling 2.5h by every 100g
Fish focuses on pectoral fin base portion injection 0.08-0.12ml colchicine；It is cut away head, the kidney of spotted maigre, it is hypotonic with KCl solution, then with admittedly
Determine liquid to fix, cell suspension, -40 DEG C of preservations are made；Cell suspension is taken, is dripped on clean slide, 60 DEG C of aging 2.5-3.5h, system
Obtain the slide of aging；
Two, spotted maigre fin ray DNA is extracted
Spotted maigre fin ray DNA is extracted with cell/tissue genome DNA extracting reagent kit；
Three, FISH probe is prepared
Spotted maigre fin ray DNA is marked with nick translation method, FISH probe length obtained is between 200-750bp；
Four, genome breeding
1) slide is denaturalized: the slide of aging is denaturalized in the formamide that 74 DEG C of the volumetric concentration now matched is 80%
2.5min, then be successively dehydrated respectively with the ethyl alcohol that mass concentration is 70%, 80%, 90%, 100%, 100%, the glass being denaturalized is made
Piece；
2) FISH probe is denaturalized: the FISH probe prepared being added in hybridization buffer HB, so that final concentration of
1.5-2ng/ μ l, 72 DEG C of denaturation 8min are placed on ice cube immediately after, and the FISH probe of denaturation is made；
3) hybridize: the FISH probe of denaturation being added on the slide being denaturalized, is sealed up with sealed membrane, in 37 DEG C of insulating boxs
Middle hybridization 12-16h；
4) amplify: being taken out after hybridization, after being placed at room temperature for 0.8-1.2h, the formamide for the use of volumetric concentration being successively 10% is molten
Liquid, 4 × SSC, 4 × SSC respectively wash 5min, there is 37 DEG C of the place incubation of FISH probe after room temperature is dried plus on AV to slide；
5) it washs: taking out after incubation, successively washed with 2*T-SSC, 4 × SSC, the dark place 4 × SSC；
6) signal detection: room temperature dry after with PI(inorganic phosphate) redyed, nail sheet for oil seal, it is then aobvious with fluorescence
Micro mirror completes microexamination and takes pictures；Red fluorescent is observed by green fluorescence filter disc group, passes through blue-fluorescence filter disc group
Green florescent signal is observed, image is shot using the charge coupling device imaging sensor that microscope is connected, utilizes cellSens
White-black pattern acquisition image is respectively adopted in software, and recombinant channel forms color image.
As a further solution of the present invention: in step 1 mixed liquor be 50g Radix Codonopsis is decocted to final volume be 50ml,
It is made after adding the BSA mixing of 0.405g NaCl and 125mg.
As a further solution of the present invention: the concentration of KCl solution is 0.075mol/l.
As a further solution of the present invention: fixer uses Ka Nuoshi fixer in step 1.
As a further solution of the present invention: cell/tissue genome DNA extracting reagent kit uses in step 2
GK0121。
As a further solution of the present invention: in step 4 volumetric concentration be 80% formamide include 12ml formamide,
20 × SSC of 1.5ml and 1.5ml aqua sterilisa.
As a further solution of the present invention: hybridization buffer HB contains hybridization basis buffer, deionization in step 4
Formamide and 50% dextran sulfate.
As a further solution of the present invention: AV is the Avidin-Alexa of final concentration of 4 μ g/ml in step 4
Fluor-488, the rabbit-anti Avidin of 488 labels.
As a further solution of the present invention: fluorescence microscope uses Olympus BX53 fluorescence microscope in step 4.
As a further solution of the present invention: the final concentration of 1 μ g/ml of PI in step 4.
Compared with prior art, the beneficial effects of the present invention are:
The present invention, which provides a kind of short-cut method, can make spotted maigre chromosome that special fluorescence distribution feature be presented, and improve yellow
The efficiency and accuracy of aunt's fish chromosome identification, solve the problems, such as that spotted maigre chromosome identification mark is poor.
Detailed description of the invention
Fig. 1 is the spotted maigre idiogram based on FISH.A. female spotted maigre FISH；B. male spotted maigre
FISH；C. female spotted maigre idiogram；D. male spotted maigre idiogram；E. female spotted maigre chromosome fluorescence
Signal 3D optical density figure；F. male spotted maigre chromosome fluorescence signal 3D optical density figure, scale=5 μm.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical scheme in the embodiment of the invention is clearly and completely described,
Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based in the present invention
Embodiment, every other embodiment obtained by those of ordinary skill in the art without making creative efforts, all
Belong to the scope of protection of the invention.
Embodiment 1
In the embodiment of the present invention, a method of display spotted maigre chromosome morphology feature, steps are as follows:
1, prepared by chromosome
The spotted maigre that weight is 400g is chosen, is injected using Three-ball method, per injection is complete temporarily to support in 25 DEG C of seawater；Preceding two
Needle focuses on pectoral fin base portion injection 0.8ml Radix Codonopsis by every 100g fish and decocts the mixed liquor of juice and BSA (Radix Codonopsis 50g is decocted to final body
Product is 50ml, and the BSA that 0.405g NaCl and 125mg is added is mixed), the second needle is injected after 15h；2.5h is in pectoral fin base before sampling
Inject 0.1ml/100g colchicine in portion；It is cut away head-kidney, it is solid with the hypotonic 40min of KCl solution of 0.075mol/l, then with Ka Nuoshi
Determine liquid to fix 3 times, -40 DEG C of preservations；Cell suspension is taken before slide denaturation, is dripped on the clean slide for having fumulus, 60 DEG C old
Change 3h.
2, spotted maigre fin ray DNA is extracted
Spotted maigre fin ray DNA is extracted with cell/tissue genome DNA extracting reagent kit (GK0121).
3, prepared by FISH probe
20 μ l of spotted maigre fin ray DNA(total volume: nick translation hybridization buffer 10 μ l, 3 μ l are marked with nick translation method
DNA(1 μ g), Polymerase polymerase 0.4 μ l(Thermo, EP0041), Dnase I(deoxyribonuclease I) 1 μ l
(wherein Dnase I 0.7mU, mU are the active unit of enzyme, i.e., enzyme amount needed for 1 micromolar substrate), residue plus aqua sterilisa
5.6 μ l)；14.5 DEG C of reaction 90min, so that FISH probe length is between 200-750bp.
4, genome breeding (FISH)
1) slide is denaturalized: formamide (the 12ml formyl for being 80% in 74 DEG C of the volumetric concentration now matched by the good slide of aging
Amine, 20 × SSC(of 1.5ml sodium citrate buffer solution), 1.5ml aqua sterilisa) in be denaturalized 2.5min, series of ethanol is dehydrated each 30s
(70%, 80%, 90%, 100%, 100%).
2) FISH probe is denaturalized: the spotted maigre genome FISH probe prepared being added to (miscellaneous in hybridization buffer HB
Hand over basis buffer, deionized formamide, 50% dextran sulfate) so that final concentration of 1.5-2ng/ μ l, 72 DEG C of denaturation 8min,
10min on ice is placed immediately after.
3) hybridize: the FISH probe being denaturalized (totally 35 μ l) being added on the slide being denaturalized, is sealed up with sealed membrane,
It is placed in magazine and hybridizes 12h in 37 DEG C of insulating boxs.
4) amplify: being taken out after 12h, be placed at room temperature for 1h, [10% formamide (10%FA), 4 × SSC, 4 × SSC] is respectively washed
5min, room temperature add the Avidin-Alexa fluor-488 of the final concentration of 4 μ g/ml of AV(of 100 μ l, the rabbits of 488 labels after drying
Anti- Avidin) to (place for having FISH probe) 37 DEG C of incubation 30min on slide；
5) it washs: being taken out after incubation, successively wash 5min with 2*T-SSC, 4 × SSC, the dark place 4 × SSC；
6) signal detection: room temperature is redyed after drying with the PI of final concentration of 1 μ g/ml, nail sheet for oil seal, after 10min
It is completed microexamination with Olympus BX53 fluorescence microscope and is taken pictures.Red fluorescence is observed by green fluorescence filter disc group (GW)
Signal observes green florescent signal by blue-fluorescence filter disc group (BW), the DP73 charge coupling device being connected using microscope
Imaging sensor (CCD) shoots image, and white-black pattern acquisition image, recombinant channel shape is respectively adopted using cellSens software
At color image.
7) image procossing: the picture taken is separated mid-term phase chromosome every of acquisition with Photoshop6.0
Come, then assists in identifying homologue with the vertical view 3D fluorescence intensity figure that Image Pro-plus6.0 obtains chromosome.
The result of embodiment 1 is as shown in Figure 1.
A, b spotted maigre male and female mid-term phase FISH of Fig. 1 are as a result, it can clearly be seen that every chromosome from a and b of Fig. 1
Fluorescence signal distribution most of also have signal distributions between end, the arm of some chromosomes.
Fig. 1 c, what d was shown is that chromosome is carried out homogenetic association simultaneously in conjunction with IPP6.0 after spotted maigre male and female PS is separated respectively
Sequence.It will be evident that the similar chromosome that can be distinguished with other homologues of signal distributions mode has 18 pairs of female, respectively
For 1,2,3,4,5,6,7,10,11,13,14,16,17,18,19,20,22, No. 24 chromosome；19 pairs of male, respectively 1,
2,3,4,5,6,7,8,11,12,13,14,15,17,18,19,20,21, No. 23 chromosome；There are notable differences for signal mode
It is similar with model mode, but be difficult to have with the chromosome that other homologues distinguish 6 pairs of female, respectively 8,9,12,15,
21, No. 23；5 pairs, respectively 9,10,21, No. 23 of male.Using IPP software, the 3D fluorescence intensity of positive signals can be with
Heterochromia is converted by fluorescence intensity difference, is facilitated eye recognition (Fig. 1 e, Fig. 1 f), it further can be by residue very
The chromosome that difficulty distinguishes combines its respective caryogram data, and spotted maigre 24 can be made close to chromosome identification pairing rate
100%。
It is obvious to a person skilled in the art that invention is not limited to the details of the above exemplary embodiments, Er Qie
In the case where without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, the present embodiments are to be considered as illustrative and not restrictive, and the scope of the present invention is by appended power
Benefit requires rather than above description limits, it is intended that all by what is fallen within the meaning and scope of the equivalent elements of the claims
Variation is included within the present invention.
In addition, it should be understood that although this specification is described in terms of embodiments, but not each embodiment is only wrapped
Containing an independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art should
It considers the specification as a whole, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
The other embodiments being understood that.
Claims (5)
1. a kind of method for showing spotted maigre chromosome morphology feature, which is characterized in that steps are as follows:
One, prepared by chromosome
Spotted maigre is chosen, is injected using Three-ball method；Preceding two needle focuses on pectoral fin base portion injection 0.6-1.0ml Radix Codonopsis by every 100g fish and decocts
The mixed liquor of juice and BSA injects the second needle after injecting the first needle 15h；Third needle is that 2.5h is heavy by every 100g fish before sampling
0.08-0.12ml colchicine is injected in pectoral fin base portion；The head-kidney for taking spotted maigre, it is hypotonic with KCl solution then solid with fixer
It is fixed, cell suspension, -40 DEG C of preservations are made；Cell suspension is taken, is dripped on clean slide, 60 DEG C of aging 2.5-3.5h, aging is made
Slide；The mixed liquor that the Radix Codonopsis decocts juice and BSA is that decoct 50g Radix Codonopsis to final volume be 50ml, is added
The BSA of 0.405g NaCl and 125mg are made after mixing；
Two, spotted maigre fin ray DNA is extracted
Spotted maigre fin ray DNA is extracted with cell/tissue genome DNA extracting reagent kit；
Three, FISH probe is prepared
Spotted maigre fin ray DNA is marked with nick translation method, FISH probe length obtained is between 200-750bp；
Four, genome breeding
1) slide is denaturalized: the slide of aging is denaturalized 2.5min in the formamide that 74 DEG C of the volumetric concentration now matched is 80%, then
It is successively dehydrated respectively with the ethyl alcohol that mass concentration is 70%, 80%, 90%, 100%, 100%, the slide being denaturalized is made；
2) FISH probe is denaturalized: the FISH probe prepared being added in hybridization buffer HB, so that final concentration of 1.5-
2ng/ μ l, 72 DEG C of denaturation 8min are placed on ice cube immediately after, and the FISH probe of denaturation is made；The hybridization buffer HB contains
There are hybridization basis buffer, deionized formamide and 50% dextran sulfate；
3) hybridize: the FISH probe of denaturation being added on the slide being denaturalized, is sealed up with sealed membrane, it is miscellaneous in 37 DEG C of insulating boxs
Hand over 12-16h；
4) amplify: being taken out after hybridization, after being placed at room temperature for 0.8-1.2h, successively use the formamide solution, 4 that volumetric concentration is 10%
× SSC, 4 × SSC respectively wash 5min, there is 37 DEG C of the place incubation of FISH probe after room temperature is dried plus on AV to slide；Wherein, AV
The rabbit-anti Avidin marked for the Avidin-Alexa fluor-488 of 4 μ g/ml；
5) it washs: taking out after incubation, successively washed with 2 × T-SSC, 4 × SSC, the dark place 4 × SSC；
6) signal detection: room temperature is redyed after drying with propidium iodide PI, then nail sheet for oil seal is completed with fluorescence microscope
Microexamination with take pictures；Red fluorescent is observed by green fluorescence filter disc group, green is observed by blue-fluorescence filter disc group
Fluorescence signal is shot image using the charge coupling device imaging sensor that microscope is connected, is distinguished using cellSens software
Image is acquired using white-black pattern, recombinant channel forms color image.
2. the method for display spotted maigre chromosome morphology feature according to claim 1, which is characterized in that KCl solution
Concentration is 0.075mol/l.
3. the method for display spotted maigre chromosome morphology feature according to claim 1, which is characterized in that solid in step 1
Liquid is determined using Ka Nuoshi fixer.
4. the method for display spotted maigre chromosome morphology feature according to claim 1, which is characterized in that body in step 4
The formamide that product concentration is 80% includes 12ml formamide, 1.5ml 20 × SSC and 1.5ml aqua sterilisa.
5. the method for display spotted maigre chromosome morphology feature according to claim 1, which is characterized in that iodine in step 4
Change the final concentration of 1 μ g/ml of the third pyridine PI.
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| CN106501040A (en) * | 2016-10-24 | 2017-03-15 | 南通大学附属医院 | Human peripheral chromosome synchronizes reagent preparation box |
| CN110411797A (en) * | 2019-07-15 | 2019-11-05 | 南通大学 | A method of reproduction cell chromosome is prepared using two step Low Osmotic Methods |
| CN112626234A (en) * | 2020-12-29 | 2021-04-09 | 集美大学 | FISH probe set of large yellow croaker chromosome and kit thereof |
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| CN104004849A (en) * | 2014-06-09 | 2014-08-27 | 南京农业大学 | Method for quickly establishing metaphase chromosome karyotype of cucumber through genomic in-situ hybridization |
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| CN104004849A (en) * | 2014-06-09 | 2014-08-27 | 南京农业大学 | Method for quickly establishing metaphase chromosome karyotype of cucumber through genomic in-situ hybridization |
Non-Patent Citations (3)
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| Assessing genetic diversity of Brazilian reef fishes by chromochromosomal and DNA markers;GALETTI PM JR等;《Genetica》;20061231;第126卷;全文 |
| 中药替代PHA体内注射获取大量鱼类肾细胞中期分裂相;容寿柏等;《淡水渔业》;19911231(第6期);6-7 |
| 大黄鱼与黄姑鱼杂交F1及其双亲的核型分析;蔡明夷等;《集美大学学报（自然科学版）》;20120930;第17卷(第5期);321-326 |
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