CN105543342B - A method of display Larimichthys crocea centromere and galianconism - Google Patents
A method of display Larimichthys crocea centromere and galianconism Download PDFInfo
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- CN105543342B CN105543342B CN201510835498.8A CN201510835498A CN105543342B CN 105543342 B CN105543342 B CN 105543342B CN 201510835498 A CN201510835498 A CN 201510835498A CN 105543342 B CN105543342 B CN 105543342B
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Abstract
The invention discloses a kind of methods for showing Larimichthys crocea centromere and galianconism, belong to molecular cytogenetics field, the molecular labeling is the one section of 28S rDNA sequence cloned from human saliva, then it is prepared into probe (being named as H-P3K), hybridize with Larimichthys crocea metaphase chromosome, to obtain positive signal.When the probe makees FISH to Larimichthys crocea chromosome, positive signal can be detected on the centromere and galianconism region of every metaphase chromosome, the signal strength of galianconism is weaker than centromere signal strength on same chromosome, and there is also differences for centromere signal strength between different chromosomes.The present invention is used to determine the position in centromere, analyzes karyotype and research chromosome structure；Positive signal can be used as the label of chromosome identification and pairing.
Description
Technical field
The invention belongs to molecular and cytogenetic techniques field, it is related to a kind of chromosomal gene localization method, specifically one
The method in kind display Larimichthys crocea centromere and galianconism.
Background technique
Larimichthys crocea (Larimichthys crocea) is commonly called as " yellow croaker ", " cucumber fish ", is under the jurisdiction of Perciformes
(Perciformes), Sciaenidae (Scieanidae), yellow croaker category (Larimichthys) are warm warm water mass migration fishes
Class is mainly distributed on China's the East China Sea, the Taiwan Straits and the South Sea, is the peculiar fingerling of China coastal seas, is four big oceans
One of economic fish.Its seedling cultivation scale, number of practitioner etc. all occupy first of marine fish.Larimichthys crocea has 48 dyes
Colour solid does not find sex chromosome.Chromosome length is shorter, and 24 pairs of chromosome sizes are successively successively decreased, difference in size between chromosome
It is not significant.So the report in relation to Larimichthys crocea karyotype formulas is inconsistent or even arm is not more identical than also, show that Larimichthys crocea chromosome can
There can be polymorphism.
Related Larimichthys crocea karyotype research report is still less, and the dye image in most reports is second-rate；
Traditional karyotyping is rested essentially within, chromosome and segment identification are still highly difficult.This status has been not suitable for current quick
The genomics of development and the research of functional gene need.Fluorescence in situ hybridization is nearly 20 years technologies to grow up, makes to dye
It is horizontal that body analysis enters molecular cytogenetics.RDNA coded sequence is most one of conserved sequence in eukaryotic gene group, is drawn
Object can be general in higher taxon.Research data shows that rDNA may be disseminated in genome, generates new rDNA
Site, changeable rDNA copy, or even be associated with some other multigene family.RDNA is to be most commonly used as visiting in FISH test
One of sequence of needle.Therefore, positioning rDNA can identify for chromosome provides mark, can also be the chromosome of research relative species
Evolution offer data.
Summary of the invention
The purpose of the present invention is to provide a kind of methods for showing Larimichthys crocea centromere and galianconism, for determining centromere
Karyotype and research chromosome structure are analyzed, to solve the problems mentioned in the above background technology in position.
To achieve the above object, the present invention provides the following technical solutions：
A method of display Larimichthys crocea centromere and galianconism include the following steps：
(1) prepared by chromosome
20h is overweighted the pan-fried juice and 18~20 μ g BSA of pectoral fin base portion injection 7.5~8.5mg Radix Codonopsis by every gram of fish body in advance
Mixed liquor is temporarily supported in 24~26 DEG C of seawater；5h injects same reagent with same method again in advance；2.5h presses every gram of fish in advance
Weight injects the colchicine of 0.4~0.6 μ g/g in pectoral fin base portion；Take renal cell, using 0.075M KCl hypotonic 35~
45min, Ka Nuoshi fixer are fixed 2~4 times, change Ka Nuoshi fixer into methanol/acetic acid mixture before drop piece；In slide table
One layer of steam is breathed out in face, away from cell suspending liquid is added dropwise at slide 1cm, is placed in air and is spontaneously dried；
(2) human genome DNA extracts
Human saliva DNA is extracted with tissue gene group DNA extraction kit to pour into physiological saline in mouth before brushing teeth,
Gargle 25~35s, spits waste collection cup, again pours into physiological saline in mouth, and firmly gargle 1min, then will carefully gargle
Liquid is spat into disposable cup, takes 1.5ml, and room temperature is centrifuged 12~18min, and centrifugal speed 8000rmp outwells supernatant, is remained
Remaining mouth epithelial cells precipitating；
(3) prepared by probe
According to the 28S rDNA primers of the mankind：18S-UF-5'-GGCACGAGACCGATAGTCAACA-3'
18S-UR-5'-ACCTGCTGCGGATATGGGTAC-3'；
Pcr amplification reaction system is 20 μ L：Each 1 μ L of 10 μM of primer, human genome DNA 0.9~1.1 μ L, 10 ×
1.8~2.2 μ L, 2.5mmol/L dNTP mixture of buffer, 1.5~1.7 μ L, 2.5U/ μ LFastful archaeal dna polymerase 0.18
~0.22 μ L adds distilled water to 20 μ L；
Using nick translation kit label probe, reaction system is 20 μ L：10 × buffer, 1.8~2.2 μ L, reagent
Enzyme mixation 2.8~3.2 μ L, dNTP 6.8~7.2 μ L, Biotin-dUTP 0.28~0.32 μ L, mankind 28S in box
0.9~1.1 μ g of rDNA amplified production adds distilled water to take 1.90~2.10 μ to 20 μ L, 14.5~15.5 DEG C of 85~95min of reaction
Agarose gel electrophoresis of the L nick translation product for 1% is analyzed；
Remaining probe solution is anhydrous with 4.5~5.5 μ L 3M sodium acetates, 9.5~10.5 μ L salmon sperm DNAs, 98~102 μ L
Ethyl alcohol is precipitated, and is dissolved in 38~42 μ L hybridization buffers after 54.5~55.5 DEG C of dryings of uncapping, probe mixed liquor is made；
(4) fluorescence in situ hybridization (FISH)
1. the pretreatment of sample：By slide carry out aging, Proteinase K, acetone treatment, be then placed in volume fraction be 78~
In 82% formamide/2 × SSC solution, 64.5~65.5 DEG C of 2.4~2.6min of denaturation, and handled with ice ethanol dehydration；
2. probe is denaturalized：Probe mixed liquor is denaturalized 7~9min in 74~76 DEG C of water-bath, is then transferred to ice rapidly
In rapidly cool down 10min or more；
3. hybridizing：The probe mixed liquor of 20 μ L denaturation is dripped on Larimichthys crocea chromosome slide, and lid sealed membrane is placed in wet dark
In box, 36.5~37.5 DEG C of 12~16h of hatching；
4. being eluted after hybridization：It is 9~11% formamide/2 × SSC and 4 that slide after hybridization, which is successively used volume fraction,
× SSC washing；Secondly, with Avidin-Alexa Fluor488 solution and probe molecule in 36~38 DEG C, wet dark environment
Middle 28~32min of specific bond is successively washed using 4 × SSC-Trixton and 4 × SSC；Telomere repeat sequence FISH positioning needs
To pass through second of specific bond, i.e., be added dropwise after BiotinylatedAnti-Avidin solution D cultivates 30min and wash on slide
It is de-, then be added dropwise after 488 solution of Avidin-Alexa Fluor cultivates 30min and elute again；Finally, being added dropwise on slide, PI is anti-to be taken off
Toner is redyed, fluorescence microscope result；
5. signal detection：It completes microexamination using Olympus BX53 fluorescence microscope and takes pictures, pass through green fluorescence
Filter disc group observes red fluorescent, observes green florescent signal by blue-fluorescence filter disc group, is connected using microscope
DP73 charge coupling device imaging sensor (CCD) shoots image, and white-black pattern is respectively adopted using cellSens software and acquires
Image, recombinant channel form color image.
As a further solution of the present invention：In the step (1), in methanol/acetic acid mixed liquor, methanol：Acetic acid
Volume ratio is 1.8~2.2:1.
As further scheme of the invention：In the step (3), the condition of pcr amplification reaction is：97.5~98.5
DEG C 4~6min of initial denaturation；97.5~98.5 DEG C of denaturation 28~32s, 58~62 DEG C of annealing 28~32s, 71~73 DEG C extend 3~
5min is recycled 35 times；9~11min of last 71~73 DEG C of extensions, 3~5 DEG C of preservations.
Compared with prior art, the beneficial effects of the invention are as follows：
The molecular labeling is the one section of 28S rDNA sequence cloned from human saliva, probe is then prepared into, with rheum officinale
Fish metaphase chromosome hybridization, to obtain positive signal.When the probe makees FISH to Larimichthys crocea chromosome, it can be contaminated in every mid-term
Positive signal is detected on the centromere and galianconism region of colour solid, and the signal strength of galianconism is weaker than centromere on same chromosome
Signal strength, there is also differences for centromere signal strength between different chromosomes.The invention can be used as dyeing with positive signal
The label of body identification and pairing；It can also show the genomic difference of Larimichthys crocea, it is miscellaneous for the two species of assistant analysis
Hand over offspring genome at.
Detailed description of the invention
Fig. 1 is positioning of the H-P3K on Larimichthys crocea female (1), male (2) chromosome of Larimichthys crocea.
In figure：(1,2) small frame lists non-telocentric chromosome, scale=5 μm.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
Embodiment
(1) prepared by chromosome
20h is overweighted pan-fried juice/20 μ g BSA mixed liquors of pectoral fin base portion injection 8mg Radix Codonopsis by every gram of fish body in advance, is temporarily supported
In 25 DEG C of seawater；5h injects same reagent with same method again in advance；2.5h overweights pectoral fin base portion by every gram of fish body in advance
Inject the colchicine of 0.5 μ g/g；Renal cell is taken, using the hypotonic 40min of 0.075M KCl, Ka Nuoshi fixer is fixed 3 times；
Changing Ka Nuoshi fixer into volume ratio before drop piece is methanol/acetic acid=2:1 mixed liquor；Breathe out one layer of steam in surface of glass slide,
According to cell suspending liquid is added dropwise at slide 1cm, it is placed in air and spontaneously dries；
(2) human genome DNA extracts
Human saliva DNA is extracted with tissue gene group DNA extraction kit (Shanghai JaRa)；Before breakfast is brushed teeth, it will give birth to
Reason salt water pours into mouth, and gargle 30s, spits waste collection cup, again pours into physiological saline in mouth, and firmly gargle 1min, so
Gargle is carefully spat into disposable cup afterwards, takes 1.5ml, room temperature is centrifuged 15min, and centrifugal rotational speed 8000rmp outwells supernatant
Liquid, remaining mouth epithelial cells precipitate, then operating procedure of the specific steps referring to kit；
(3) prepared by probe
According to the 28S rDNA primers of the mankind：18S-UF-5'-GGCACGAGACCGATAGTCAACA-3'
18S-UR-5'-ACCTGCTGCGGATATGGGTAC-3'；
Amplification reaction system is 20 μ L：Each 1 μ L of 10 μM of primer, 1 μ L, 10 × buffer2 μ L of human genome DNA,
1.6 μ L, 2.5U/ μ LFastful archaeal dna polymerase of 2.5mmol/L dNTP mixture, 0.2 μ L, adds distilled water to 20 μ L；
Pcr amplification reaction condition is：98 DEG C of initial denaturation 5min；98 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C extend
4min is recycled 35 times；Last 72 DEG C of extensions 10min, 4 DEG C of preservations；
Using nick translation kit (Roche) label probe, reaction system is 20 μ L：10 × buffer, 2 μ L, reagent
37 μ L, Biotin-dUTP of μ L, dNTP of enzyme mixation 0.3 μ L, 1 μ g of mankind 28S rDNA amplified production in box add distilled water
To 20 μ L, 15 DEG C of reaction 90min, agarose gel electrophoresis of L mouthfuls of the 2 μ translation products for 1% is taken to analyze；
5 μ L 3M sodium acetates of remaining 18 μ L probe solution, 10 μ L salmon sperm DNAs, 100 μ L dehydrated alcohols precipitating, 55 DEG C are opened
It is dissolved in 40 μ L hybridization buffers after lid is dry, probe mixed liquor is made；
(4) fluorescence in situ hybridization (FISH)
1. the pretreatment of sample
Slide is subjected to aging, Proteinase K, acetone treatment, is then placed in formamide/2 × SSC that volume fraction is 80%
In solution, 65 DEG C of denaturation 2.5min, and rapidly with serial ice ethanol dehydration；
2. probe is denaturalized
Probe mixed liquor is denaturalized 8min in 75 DEG C of water-bath, be then transferred to rapidly in ice rapidly cool down 10min with
On；
3. hybridizing
The probe mixed liquor of 20 μ L denaturation is dripped on Larimichthys crocea chromosome slide, and lid sealed membrane is placed in wet magazine, 37
DEG C incubated overnight, brooding time are controlled in 12-16h；
4. being eluted after hybridization
Slide after hybridization is successively washed with the formamide/2 × SSC and 4 × SSC that volume fraction is 10%；Secondly, with
488 solution of Avidin-Alexa Fluor and probe molecule the specific bond 30min in 37 DEG C, wet, dark environment,
Successively washed using 4 × SSC-Trixton and 4 × SSC；Telomere repeat sequence FISH positioning is needed by second of special knot
It closes, i.e., is added dropwise after BiotinylatedAnti-Avidin solution D cultivates 30min and elutes on slide, then Avidin- is added dropwise
488 solution of AlexaFluor elutes again after cultivating 30min；It is redyed finally, PI anti-color fading agent is added dropwise on slide, fluorescence microscopy
Sem observation result；
5. signal detection
It completes microexamination using Olympus BX53 fluorescence microscope and takes pictures, seen by green fluorescence filter disc group (GW)
Red fluorescent is examined, green florescent signal is observed by blue-fluorescence filter disc group (BW), the DP73 electricity being connected using microscope
Lotus coupled device imaging sensor (CCD) shoots image, and white-black pattern acquisition image is respectively adopted using cellSens software, then
It combines channel and forms color image.
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.Any reference signs in the claims should not be construed as limiting the involved claims.
SEQUENCE LISTING
<110>Collects The American University
<120>A method of display Larimichthys crocea centromere and galianconism
<130> 2018
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 22
<212> DNA
<213>It is artificial synthesized
<400> 1
ggcacgagac cgatagtcaa ca 22
<210> 2
<211> 21
<212> DNA
<213>It is artificial synthesized
<400> 2
acctgctgcg gatatgggta c 21
<210> 3
<211> 2221
<212> DNA
<213>It is artificial synthesized
<400> 3
cctgctgcgg atatgggtat ggccctgtgc gagacttaca cagtctcccc tggattttca 60
aaggccagcc agagctcacc agatgccgcc agaaccacga cgctttccaa ggtacaggcc 120
cctctctcag ggcgaaccca ttccagggcg tcctgccctt cacaaagaaa agagaaccct 180
ccccggggct cctgcatgct tctccaggat cggtcacatt accgcactgg aagcctcatg 240
gtgcccatct ctgccactct ggattcaggg atctgaaccc gtctcccttt caaccggctg 300
agggcaatgg aggccctcac ctgtcccttc ggaacggcac tcacccatct ctcaggacca 360
accgacacat gttcaactgt tgttcacatg gaacccttct ccacttaggc cttcaaagtt 420
ctcatttgaa aatttgctac taccaccaag ttctgcacct gcagtggctc caaccgggcc 480
catgccctag acttcaaagc ttaccgcagc ggccctccta ctcgtcgcgg catagaatcc 540
gtggggctct gggggagggt gcaggagagg acccccacca cactcacacg ggcataacgc 600
ccctgccgct cccgtccact ctcaactgcc agcaacggcc gggtatgggc cggacgctcc 660
agcaccatcc attttcaggg ctagttgatt cagcaggtga gttgttacac actccttagc 720
agattccaac ttccatggcc accgtcctgc tgtctatatc aaccaacaca ttttctcggg 780
tctgatgatt gttggcatcg ggcaccttaa cccggcattc tgttcatccc acaatgccag 840
ttctacttac caaaagtagc ccactaagca ctcgcattcc acgcccagct ccacgccagc 900
aagccggact tcttacacat ttaaagtttc agaataggtt gagatcgttt cggcaccaag 960
acctctaatc attcgcttta ccggataaaa ctgtgtgggg aggttaggtt tgcgagagca 1020
ccaggtatcc taagggaaac ttcagaggga accagctact agatggtttg attagtcttt 1080
taccccctat acccaggtcg aaagaccgat ttgcacgtca gggccgctac ggacctccac 1140
cagcgtttcc tctggttcca ccctgcccag gcatagttca ccatcttttg ggtcctaaca 1200
cgtgcgctcg tgctccacct ccctgccgcg gcaggcgaga cgggacggtg gtatgccgtc 1260
ggcagactgg agaggcctcg ggatcccacc tcggccagcg agcacaccgc ccttcacctt 1320
cattgctcca cagcggcttt cgtgggagcc cctgactcac caacgtgtta accttggtcc 1380
atttttcaag tagggtcggg tggggagaca acgtcgccgc tgaccccatg tgctcgctcc 1440
accgagtgac gtccgcatgg ccccgagaga actcccccgg ggccgacagt gtgacccgcc 1500
catggcacac tggggacagt ctaccccacc cctctatgcg gtcccccgtc ggtggggatg 1560
catggaggcg ggtgggagag cagtcacacc gtgggcgggg tggcccagcc ccccaccagg 1620
agacacaggc gcacccccaa gggggtgggc accggcaagg gagagcacgg caatgggtct 1680
cgctccctcc gccctgggat ttggctaggg ctgctgcggg gggtctgtaa ctcaggaggt 1740
tcggtcccac cactgccgcc accccgaccc gcatggcctc ccgagggagg acgcggggcc 1800
ggggagaaga cggggaagga cggacggggc ccccagaccc acctttcccc accgggcctt 1860
cccaaccatc ccagagccag ccacagtgca ccgctgtggt ggaaatgcgc cgggcggccg 1920
ccagtcgcca gttaggggat ggtcacccac cgaccccggc cctgcccgcc cacaccaacc 1980
cacctccggg gaggaggagg ggcagtcggg gagggagggc gggtggaggg ttcgggagga 2040
acttggggcg ggaaagatcc actgggccac cgacacggcc ggacccgcca ccaggttgaa 2100
tcctctgagc agactgcgcg gaccccaccc gtttacctct taatggtttc acgccctctt 2160
gcacgctctt caaagttctt ttcaactttc ccttacggta cttcactatc ggtctcgtgc 2220
c 2221
Claims (3)
1. a kind of method for showing Larimichthys crocea centromere and galianconism, which is characterized in that include the following steps：
(1) prepared by chromosome
20h overweights the pan-fried juice of pectoral fin base portion injection 7.5~8.5mg Radix Codonopsis by every gram of fish body in advance and 18~20 μ g BSA are mixed
Liquid is temporarily supported in 24~26 DEG C of seawater；5h injects same reagent with same method again in advance；2.5h presses every gram of fish body weight in advance
The colchicine of 0.4~0.6 μ g/g is injected in pectoral fin base portion；Renal cell is taken, using the hypotonic 35~45min of 0.075M KCl,
Ka Nuoshi fixer is fixed 2~4 times, changes Ka Nuoshi fixer into methanol/acetic acid mixture before drop piece；Breathe out one in surface of glass slide
Layer steam is placed in air and spontaneously dries away from cell suspending liquid is added dropwise at slide 1cm；
(2) human genome DNA extracts
Human saliva DNA is extracted with tissue gene group DNA extraction kit to pour into physiological saline in mouth before brushing teeth, and is gargled
25~35s spits waste collection cup, again pours into physiological saline in mouth, and firmly gargle 1min, then carefully spits gargle
Enter in disposable cup, take 1.5ml, room temperature is centrifuged 12~18min, centrifugal speed 8000rpm, outwells supernatant, remaining mouth
Chamber epithelial cell precipitating；
(3) prepared by probe
According to the 28S rDNA primers of the mankind：18S-UF-5'-GGCACGAGACCGATAGTCAACA-3'18S-UR-
5'-ACCTGCTGCGGATATGGGTAC-3'；
Pcr amplification reaction system is 20 μ L：Each 1 μ L of 10 μM of primer, human genome DNA 0.9~1.1 μ L, 10 × buffer
1.8~2.2 μ L, 2.5mmol/L dNTP mixture, 1.5~1.7 μ L, 2.5U/ μ LFastful archaeal dna polymerase, 0.18~0.22 μ
L adds distilled water to 20 μ L；
Using nick translation kit label probe, reaction system is 20 μ L：10 × buffer, 1.8~2.2 μ L, in kit
Enzyme mixation 2.8~3.2 μ L, dNTP 6.8~7.2 μ L, Biotin-dUTP 0.28~0.32 μ L, mankind 28S rDNA expands
Increase production 0.9~1.1 μ g of object, distilled water is added to take 1.90~2.10 μ L notches to 20 μ L, 14.5~15.5 DEG C of 85~95min of reaction
The agarose gel electrophoresis that product is translated for 1% is analyzed；
Remaining probe solution 4.5~5.5 μ L 3M sodium acetates, 9.5~10.5 μ L salmon sperm DNAs, 98~102 μ L dehydrated alcohols
It is precipitated, is dissolved in 38~42 μ L hybridization buffers after 54.5~55.5 DEG C of dryings of uncapping, probe mixed liquor is made；
(4) fluorescence in situ hybridization
1. the pretreatment of sample：Slide is subjected to aging, Proteinase K, acetone treatment, being then placed in volume fraction is 78~82%
Formamide/2 × SSC solution in, 64.5~65.5 DEG C of 2.4~2.6min of denaturation, and with ice ethanol dehydration handle；
2. probe is denaturalized：Probe mixed liquor is denaturalized 7~9min in 74~76 DEG C of water-bath, is then transferred to rapidly fast in ice
Quickly cooling but 10min or more；
3. hybridizing：The probe mixed liquor of 20 μ L denaturation is dripped on Larimichthys crocea chromosome slide, and lid sealed membrane is placed in wet magazine
In, 36.5~37.5 DEG C of 12~16h of hatching；
4. being eluted after hybridization：It is 9~11% formamide/2 × SSC and 4 × SSC that slide after hybridization, which is successively used volume fraction,
Washing；Secondly, special in 36~38 DEG C, wet dark environment with Avidin-Alexa Fluor488 solution and probe molecule
Different 28~32min of combination is successively washed using 4 × SSC-Trixton and 4 × SSC；Telomere repeat sequence FISH positioning need through
Second of specific bond is crossed, i.e., is added dropwise after BiotinylatedAnti-Avidin solution D cultivates 30min and elutes on slide,
It is added dropwise after 488 solution of Avidin-Alexa Fluor cultivates 30min and elutes again again；Finally, PI anti-color fading agent is added dropwise on slide
It redyes, fluorescence microscope result；
5. signal detection：It completes microexamination using Olympus BX53 fluorescence microscope and takes pictures, pass through green fluorescence filter disc
Group observation red fluorescent, observes green florescent signal by blue-fluorescence filter disc group, the DP73 electricity being connected using microscope
Lotus coupled device imaging sensor (CCD) shoots image, and white-black pattern acquisition image is respectively adopted using cellSens software, then
It combines channel and forms color image.
2. the method in display Larimichthys crocea centromere and galianconism according to claim 1, which is characterized in that the step (1)
In, in methanol/acetic acid mixed liquor, methanol：The volume ratio of acetic acid is 1.8~2.2:1.
3. the method in display Larimichthys crocea centromere and galianconism according to claim 1, which is characterized in that the step (3)
In, the condition of pcr amplification reaction is：97.5~98.5 DEG C of 4~6min of initial denaturation；97.5~98.5 DEG C of denaturation 28~32s, 58~
62 DEG C of annealing 28~32s, 71~73 DEG C of 3~5min of extension are recycled 35 times；Last 71~73 DEG C of extensions 9~11min, 3~5 DEG C
It saves.
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| CN201510835498.8A CN105543342B (en) | 2015-11-26 | 2015-11-26 | A method of display Larimichthys crocea centromere and galianconism |
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| CN201510835498.8A CN105543342B (en) | 2015-11-26 | 2015-11-26 | A method of display Larimichthys crocea centromere and galianconism |
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| CN105543342A CN105543342A (en) | 2016-05-04 |
| CN105543342B true CN105543342B (en) | 2018-11-23 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974622A (en) * | 2010-09-28 | 2011-02-16 | 大连海洋大学 | Fluorescence in situ hybridization (FISH) method for fish chromosomes |
| CN102787166A (en) * | 2012-07-16 | 2012-11-21 | 北京林业大学 | Fluorescence in situ hybridization method of prunus mume chromosome |
| CN104073568A (en) * | 2014-07-18 | 2014-10-01 | 西南大学 | Fluorescence in situ hybridization method for metaphase chromosome of mulberry |
| CN104120171A (en) * | 2013-04-25 | 2014-10-29 | 中国疾病预防控制中心辐射防护与核安全医学所 | Radiation induced cell chromosome morphology detection method |
-
2015
- 2015-11-26 CN CN201510835498.8A patent/CN105543342B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974622A (en) * | 2010-09-28 | 2011-02-16 | 大连海洋大学 | Fluorescence in situ hybridization (FISH) method for fish chromosomes |
| CN102787166A (en) * | 2012-07-16 | 2012-11-21 | 北京林业大学 | Fluorescence in situ hybridization method of prunus mume chromosome |
| CN104120171A (en) * | 2013-04-25 | 2014-10-29 | 中国疾病预防控制中心辐射防护与核安全医学所 | Radiation induced cell chromosome morphology detection method |
| CN104073568A (en) * | 2014-07-18 | 2014-10-01 | 西南大学 | Fluorescence in situ hybridization method for metaphase chromosome of mulberry |
Non-Patent Citations (5)
| Title |
|---|
| 东海区常见养殖鱼类染色体核型研究;王晓艳;《中国优秀硕士学位论文全文数据库 农业科技辑》;20120915(第9期);D052-19 * |
| 大黄鱼染色体核型研究;全成干等;《厦门大学学报(自然科学版)》;20000131;第39卷(第1期);第107-110页 * |
| 美国红鱼(Sciaenops ocellata)的核型、Ag-NORs和C-带分析;王晓艳等;《海洋与湖沼》;20120131;第43卷(第1期);第84-87页 * |
| 荧光原位杂交技术在鱼类基因定位研究中的应用;鲁翠云等;《水产学杂志》;20130831;第26卷(第4期);第51-57页 * |
| 青岛文昌鱼(Branchiostoma belcheri tsingtauense)染色体荧光原位杂交条件研究;赵婷婷等;《海洋与湖沼》;20121130;第43卷(第6期);第1187-1191页 * |
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