CN108441539A - A method of using few sequence detection forest chromosome end - Google Patents
A method of using few sequence detection forest chromosome end Download PDFInfo
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- CN108441539A CN108441539A CN201810319640.7A CN201810319640A CN108441539A CN 108441539 A CN108441539 A CN 108441539A CN 201810319640 A CN201810319640 A CN 201810319640A CN 108441539 A CN108441539 A CN 108441539A
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- 238000000034 method Methods 0.000 title claims abstract description 43
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- 241000972773 Aulopiformes Species 0.000 claims abstract description 13
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- 238000007901 in situ hybridization Methods 0.000 claims abstract description 11
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 13
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- 238000002156 mixing Methods 0.000 claims description 5
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- 101100008044 Caenorhabditis elegans cut-1 gene Proteins 0.000 claims description 3
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- 102000004190 Enzymes Human genes 0.000 claims description 3
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- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical class [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims 1
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- 235000005979 Citrus limon Nutrition 0.000 description 2
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- 238000007689 inspection Methods 0.000 description 2
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- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 108010059892 Cellulase Proteins 0.000 description 1
- 241000219766 Erythrina Species 0.000 description 1
- 240000006212 Erythrina crista-galli Species 0.000 description 1
- 241000124701 Litsea baviensis Species 0.000 description 1
- 241000712776 Litsea elongata Species 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The present invention provides a kind of method using few sequence detection forest chromosome end.This method passes through (AGGGTTT)3Few sequence probe and kit comprising the probe are realized, described (AGGGTTT)3The base sequence of few sequence probe is：5 ' AGGGTTTAGGGTTTAGGGTTT 3 ' for identification and mark forest chromosome end；Kit includes：Probe；Enzymolysis liquid；20 × SSC buffer solutions；2 × SSC buffer solutions；70%FA liquid；50%DS liquid；Salmon sperm dna；Method includes:Obtain forest Chromosome glass slide sample；Denaturation；Fluorescence in situ hybridization；Signal detection；Recycle glass slide.The present invention method can Reusability film-making, and probe prepare be easy, quickly, high sensitivity, the probe only for chromosome end few sequence, and tip of a root film-making be easy, facilitate acquisition metaphase chromosome, the entire experiment flow time is short, easy to operate.
Description
Technical field
The invention belongs to the application fields of fluorescence in situ hybridization probe, and in particular to a kind of to be contaminated using few sequence detection forest
The method of colour solid end.
Background technology
Currently, the method for detection forest chromosome is mostly simple microscopic observation, carries out photo and analyze, forest chromosome
It is relatively herbal to want small, many times the number of chromosome, size and form all cannot be distinguished clearly, cause reality
The significant errors tested.
Fluorescence in situ hybridization technique (fluorescence in situ hybridization, FISH) is in radioactivity original
A kind of on-radiation molecular cytogenetics technology to grow up on the basis of the hybridization technique of position replaces isotope mark with fluorescent marker
A kind of new in-situ hybridization method remembered and formed.FISH can more intuitively observe forest chromosome, efficiently and quick, but
It is to carry out fluorescent marker to chromosome to become difficult point therein.The simple and quick side of fluorescent marker is carried out to chromosome in addition to lacking
Outside method, there is also required target dna quantity in labeling process is more, thereby increases and it is possible to and marked erroneous can be caused, label is duplicated,
Lead to problems such as chromosome number imperfect, unintelligible.
The present invention establishes and stablizes easily by designing, synthesizing the few sequence probe for detecting forest chromosome end
Fluorescence in situ hybridization reaction system overcomes the problems of existing forest chromosome detection, is the inspection of forest tree chromosome
It surveys and new method is provided.
Invention content
In view of the problems of the existing technology, the present invention provides a kind of side using few sequence detection forest chromosome end
Method.The technical scheme is that：
A method of using few sequence detection forest chromosome end, this method passes through (AGGGTTT)3Few sequence probe
And the kit comprising the probe is realized, it is described (AGGGTTT)3The base sequence of few sequence probe is：5′-
AGGGTTTAGGGTTTAGGGTTT-3 ' for identification and marks forest chromosome end；The kit includes：(1) described
Probe；(2) enzymolysis liquid；(3) 20 × SSC buffer solutions；(4) 2 × SSC buffer solutions；(5) 70%FA liquid；(6) 50%DS liquid；(7) salmon
Milt DNA；It the described method comprises the following steps：
1) forest Chromosome glass slide sample is obtained：Clip tree seed new life 1.0~1.5cm organizations of root tips, are used successively
Mixture of ice and water processing 18~for 24 hours, glacial acetic acid handle 5min, after being cleaned with distilled water, cut 1~2mm of Root apical meristem, set
In enzymolysis liquid 45~60min is digested in 37 DEG C；
Enzymolysis liquid is removed, cleans Root apical meristem successively with distilled water, 70%, 90%, 100% alcohol successively, then add
Enter glacial acetic acid and organization of root tips suspension is made, drips on glass slide, dry, microscopy, the load glass of chromosome will be clearly observable
Piece is marked, and is placed in -20 DEG C of storage boxes and preserves；
2) it is denaturalized：The 1.5mL centrifuge tubes of sterilizing are got out, often 2 μ L salmon sperm dnas are added in pipe, then 0.5 μ L probes add
Enter 10 μ L100%FA, 2 20 × SSC of μ L and 4 μ L50%DS.Brief centrifugation after mixing is denaturalized 10min, immediately in boiling water bath
It is placed in and places 20min on ice, prevent its renaturation；
3) fluorescence in situ hybridization：Glass slide in step 1) 2 × SSC buffer solutions are handled twice, each 5min, then according to
Secondary 70% alcohol, 90% alcohol, the processing of 100% alcohol gradient, each 5min, room temperature are added dropwise 70%FA liquid after drying, cover
Coverslip handles 2min in 80 DEG C；
It is handled successively with 70% alcohol, 90% alcohol, 100% alcohol gradient later, 10 μ L are added dropwise in each 5min after drying
Glass slide is placed 1.5~2h by the denaturation mixed liquor of step 2) in 37 DEG C of insulating boxs under shading, successively with 2 × SSC buffer solutions
3min, distilled water cleaning 3min are cleaned, is finally dried at normal temperatures；
4) signal detection：DAPI is added on glass slide after drying, is detected with fluorescence microscope, and acquisition testing figure
Picture；
5) glass slide is recycled：Glass slide is placed in 2 × SSC buffer solutions, 60 DEG C of water-bath 10min, with 70%, 90%,
100% alcohol handles 10min respectively, is placed under fluorescent lamp later for 24 hours, and centralized collection is placed in -20 DEG C of storage boxes, in case
Next time uses.
Further, the probe marks the 5 ' of its sequence to hold using FAM.
Further, the preparation method of the enzymolysis liquid is：Per 10mLddH22g cellulases and 1g pectin are added in O
Enzyme.
Further, the preparation method of 20 × SSC buffer solutions is：DdH per 1000mL288.23g lemons are added in O
Sour sodium and 175.32g sodium chloride.
Further, the preparation method of 2 × SSC buffer solutions is：20 × SSC buffer solutions are diluted 10 times.
Further, the 70%FA liquid by deionized formamide (FA) and 2 × SSC buffer solutions according to volume ratio 7:3 groups
At.
Further, the preparation method of the 50%DS liquid is：Every 500 μ L ddH20.5g dextran sulfates are added in 0,
Overnight in 37 DEG C of concussions, the brief centrifugation mixing after complete melt.
Further, the processing method of the salmon sperm dna is：By the salmon sperm dna dissolved at room temperature (10mg/mL) height
Sterilization treatment 10min is pressed, boiling water, which boils, makes its DNA be denaturalized 10min, -20 DEG C of preservations.
The beneficial effects of the present invention are：The present invention is to utilize a kind of use on the basis of fluorescence in situ hybridization technique
Probe in detection forest chromosome end, the probe mainly identify that the few sequence of forest chromosome end, help are established more
Stablize convenient chromosome fluorescence in-situ hybridization reaction system.The method of the present invention is applied to the inspection of detection forest chromosome end
It surveys, the reagent composition in used kit is simpler, and it is convenient to prepare, and has easy to operate it can be readily appreciated that and repeating
The good advantage of property.The present invention method can Reusability film-making, and probe prepare be easy, quickly, high sensitivity, the probe is only
For the few sequence of chromosome end, and tip of a root film-making is easy, and facilitates acquisition metaphase chromosome, and the entire experiment flow time is short,
It is easy to operate.
Description of the drawings
Fig. 1 is (AGGGTTT) of the invention3The signal site distribution map of the Croton tiglium chromosomes of probe label
Picture；(AGGGTTT)3Probe is held using FAM labels 5 ', shows that hybridization signal green as shown in the figure, scale are 3 μm.
Fig. 2 is (AGGGTTT) of the invention3The signal position of the Erythrina crista-galli chromosomes of probe label
Point distributed image；(AGGGTTT)3Probe is held using FAM labels 5 ', shows hybridization signal green as shown in the figure, scale 3
μm。
Fig. 3 is (AGGGTTT) of the invention3The signal site distribution of the Litseabaviensis chromosomes of probe label
Image；(AGGGTTT)3Probe is held using FAM labels 5 ', shows that hybridization signal green as shown in the figure, scale are 3 μm.
Fig. 4 is (AGGGTTT) of the invention3The signal site distribution map of the Litseaelongata chromosomes of probe label
Picture；(AGGGTTT)3Probe is held using FAM labels 5 ', shows that hybridization signal green as shown in the figure, scale are 3 μm.
Fig. 5 is (AGGGTTT) of the invention3The signal position of the Podocarpusmacrophyllus chromosomes of probe label
Point distributed image；(AGGGTTT)3Probe is held using FAM labels 5 ', green as shown in the figure, and scale is 5 μm.
Fig. 6 is (AGGGTTT) of the invention3The signal site of the Quercusaquifolioides chromosomes of probe label
Distributed image；(AGGGTTT)3Probe is held using FAM labels 5 ', green as shown in the figure, and scale is 5 μm.
Fig. 7 is (AGGGTTT) of the invention3The signal site of the Robiniapseudoacacia chromosomes of probe label
Distributed image, (AGGGTTT)3Probe is held using FAM labels 5 ', green as shown in the figure, and scale is 3 μm.
Specific implementation mode
Probe is synthesized by Beijing Sheng Gong companies used by the present invention is implemented, and the probe of synthesis is diluted to 100M with 1 × TE
Concentration be stored in -20 DEG C, probe face concentration needs to be diluted to 20M again, also in -20 DEG C preservation.
Forest material is shown in Table 1 used by the present invention is implemented.
Forest species material used by 1 present invention of table is implemented
The present invention is described in further details with specific embodiment below in conjunction with the accompanying drawings, described is the solution to the present invention
It releases rather than limits.
The specific embodiment of the invention provides a kind of method using few sequence detection forest chromosome end, and this method passes through
(AGGGTTT)3Few sequence probe and kit comprising the probe are realized, described (AGGGTTT)3The base sequence of few sequence probe
It is classified as：5 '-AGGGTTTAGGGTTTAGGGTTT-3 ' for identification and mark forest chromosome end, its sequence are marked using FAM
5 ' ends of row；The kit includes：(1) probe；(2) enzymolysis liquid；Preparation method is：Per 10mLddH22g is added in O
Cellulase and 1g pectases；(3) 20 × SSC buffer solutions；Preparation method is：DdH per 1000mL288.23g lemons are added in O
Sour sodium and 175.32g sodium chloride；(4) 2 × SSC buffer solutions；Preparation method is：20 × SSC buffer solutions are diluted 10 times；(5)
70%FA liquid；By deionized formamide (FA) and 2 × SSC buffer solutions according to volume ratio 7:3 compositions；(6) 50%DS liquid；Preparation side
Method is：Every 500 μ LddH20.5g dextran sulfates are added in 0, overnight in 37 DEG C of concussions, brief centrifugation is mixed after complete melt
It is even；(7) salmon sperm dna；The processing method of the salmon sperm dna is：By the salmon sperm dna dissolved at room temperature (10mg/mL) height
Sterilization treatment 10min is pressed, boiling water, which boils, makes its DNA be denaturalized 10min, -20 DEG C of preservations.This approach includes the following steps：
1) forest Chromosome glass slide sample：Clip tree seed new life 1.0~1.5cm organizations of root tips, use ice water successively
Mixture processing 18~for 24 hours, glacial acetic acid handle 5min, after being cleaned with distilled water, cut 1~2mm of Root apical meristem, be placed in enzyme
It solves in liquid and digests 45~60min in 37 DEG C；
Enzymolysis liquid is removed, cleans Root apical meristem successively with distilled water, 70%, 90%, 100% alcohol successively, then add
Enter glacial acetic acid and organization of root tips suspension is made, drips on glass slide, dry, microscopy, the load glass of chromosome will be clearly observable
Piece is marked, and is placed in -20 DEG C of storage boxes and preserves；
2) it is denaturalized：The 1.5mL centrifuge tubes of sterilizing are got out, often 2 μ L salmon sperm dnas are added in pipe, then 0.5 μ L probes add
Enter 10 μ L 100%FA, 2 20 × SSC of μ L and 4 μ L 50%DS.Brief centrifugation after mixing, is denaturalized 10min in boiling water bath, stands
It is placed in and places 20min on ice, prevent its renaturation；
3) fluorescence in situ hybridization：Glass slide in step 1) 2 × SSC buffer solutions are handled twice, each 5min, then according to
Secondary 70% alcohol, 90% alcohol, the processing of 100% alcohol gradient, each 5min, room temperature are added dropwise 70%FA liquid after drying, cover
Coverslip handles 2min in 80 DEG C；
It is handled successively with 70% alcohol, 90% alcohol, 100% alcohol gradient later, 10 μ L are added dropwise in each 5min after drying
Glass slide is placed 1.5~2h by the denaturation mixed liquor of step 2) in 37 DEG C of insulating boxs under shading, successively with 2 × SSC buffer solutions
3min, distilled water cleaning 2min are cleaned, is finally dried at normal temperatures；
4) signal detection：DAPI is added on glass slide after drying, is detected with fluorescence microscope, and acquisition testing figure
Picture；
5) glass slide is recycled：Glass slide is placed in 2 × SSC buffer solutions, 60 DEG C of water-bath 10min, with 70%, 90%,
100% alcohol handles 10min respectively, is placed under fluorescent lamp later for 24 hours, and centralized collection is placed in -20 DEG C of storage boxes, in case
Next time uses.
Embodiment 1
The present embodiment uses (AGGGTTT)3Probe and its kit mark Croton tiglium to contaminate by the above method
Colour solid, by fluorescence microscope it is observed that green hybridization signal, the scale in figure is 3 μm.
Embodiment 2
The present embodiment uses (AGGGTTT)3Probe and its kit mark Erythrina crista- by the above method
Galli chromosomes, by fluorescence microscope it is observed that green hybridization signal, the scale in figure is 3 μm.
Embodiment 3
The present embodiment uses (AGGGTTT)3Probe and its kit are contaminated by above method label L itseabaviensis
Colour solid, by fluorescence microscope it is observed that green hybridization signal, the scale in figure is 3 μm.
Embodiment 4
The present embodiment uses (AGGGTTT)3Probe and its kit are contaminated by above method label L itseaelongata
Colour solid, by fluorescence microscope it is observed that green hybridization signal, the scale in figure is 3 μm.
Embodiment 5
The present embodiment uses (AGGGTTT)3Probe and its kit are marked by the above method
Podocarpusmacrophyllus chromosomes, by fluorescence microscope it is observed that green hybridization signal, the scale in figure
It is 3 μm.
Embodiment 6
The present embodiment uses (AGGGTTT)3Probe and its kit are marked by the above method
Quercusaquifolioides chromosomes, by fluorescence microscope it is observed that green hybridization signal, the scale in figure is 3
μm。
Embodiment 7
The present embodiment uses (AGGGTTT)3Probe and its kit are marked by the above method
Robiniapseudoacacia chromosomes, by fluorescence microscope it is observed that green hybridization signal, the scale in figure is 3 μ
m。
The method of the specific embodiment of the invention can Reusability film-making, and probe prepare be easy, quickly, high sensitivity,
Probe is only for the few sequence of forest chromosome end, and tip of a root film-making is easy, and facilitates acquisition metaphase chromosome, entire experiment stream
The journey time is short, easy to operate.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, changes, replacing and modification.
Sequence table
<110>Sichuan Agricultural University
<120>A method of using few sequence detection forest chromosome end
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
agggtttagg gtttagggtt t 21
Claims (8)
1. a kind of method using few sequence detection forest chromosome end, which is characterized in that this method passes through (AGGGTTT)3It is few
Sequence probes and kit comprising the probe are realized, described (AGGGTTT)3The base sequence of few sequence probe is：5′-
AGGGTTTAGGGTTTAGGGTTT-3 ' for identification and marks forest chromosome end；The kit includes：(1) described
Probe；(2) enzymolysis liquid；(3) 20 × SSC buffer solutions；(4) 2 × SSC buffer solutions；(5) 70%FA liquid；(6) 50%DS liquid；(7) salmon
Milt DNA；It the described method comprises the following steps：
1) forest Chromosome glass slide sample is obtained：Clip tree seed new life 1.0~1.5cm organizations of root tips, use ice water successively
Mixture processing 18~for 24 hours, glacial acetic acid handle 5min, after being cleaned with distilled water, cut 1~2mm of Root apical meristem, be placed in enzyme
It solves in liquid and digests 45~60min in 37 DEG C；
Enzymolysis liquid is removed, Root apical meristem is cleaned successively with distilled water, 70%, 90%, 100% alcohol successively, adds ice
Acetic acid is made organization of root tips suspension, drips on glass slide, dries, microscopy, will be clearly observable the glass slide of chromosome into
Line flag is placed in -20 DEG C of storage boxes and preserves；
2) it is denaturalized：The 1.5mL centrifuge tubes of sterilizing are got out, often 2 μ L salmon sperm dnas are added in pipe, then 10 μ are added in 0.5 μ L probes
L100%FA, 2 20 × SSC of μ L and 4 μ L50%DS.Brief centrifugation after mixing, is denaturalized 10min in boiling water bath, is immediately placed on ice
Upper placement 20min, prevents its renaturation；
3) fluorescence in situ hybridization：Glass slide in step 1) 2 × SSC buffer solutions are handled twice, each 5min, then used successively
70%FA liquid is added dropwise after drying in 70% alcohol, 90% alcohol, the processing of 100% alcohol gradient, each 5min, room temperature, and close the lid glass
Piece handles 2min in 80 DEG C；
It is handled successively with 70% alcohol, 90% alcohol, 100% alcohol gradient later, 10 μ L steps are added dropwise in each 5min after drying
2) glass slide is placed 1.5~2h by denaturation mixed liquor in 37 DEG C of insulating boxs under shading, successively with 2 × SSC buffer solution for cleaning
3min, distilled water clean 3min, finally dry at normal temperatures；
4) signal detection：DAPI is added on glass slide after drying, is detected with fluorescence microscope, and acquisition testing image；
5) glass slide is recycled：Glass slide is placed in 2 × SSC buffer solutions, 60 DEG C of water-bath 10min, with 70%, 90%, 100% wine
Essence handles 10min respectively, is placed under fluorescent lamp later for 24 hours, centralized collection is placed in -20 DEG C of storage boxes, in case next time makes
With.
2. a kind of method using few sequence detection forest chromosome end according to claim 1, which is characterized in that institute
State probe marks the 5 ' of its sequence to hold using FAM.
3. a kind of method using few sequence detection forest chromosome end according to claim 1, which is characterized in that institute
The preparation method for stating enzymolysis liquid is：Per 10mLddH22g cellulases and 1g pectases are added in O.
4. a kind of method using few sequence detection forest chromosome end according to claim 1, which is characterized in that institute
The preparation method for stating 20 × SSC buffer solutions is：DdH per 1000mL288.23g sodium citrates and 175.32g sodium chloride is added in O.
5. a kind of method using few sequence detection forest chromosome end according to claim 4, which is characterized in that institute
The preparation method for stating 2 × SSC buffer solutions is：20 × SSC buffer solutions are diluted 10 times.
6. a kind of method using few sequence detection forest chromosome end according to claim 1 or 4, feature exist
In the 70%FA liquid is by deionized formamide (FA) and 2 × SSC buffer solutions according to volume ratio 7:3 compositions.
7. a kind of method using few sequence detection forest chromosome end according to claim 1, which is characterized in that institute
The preparation method for stating 50%DS liquid is：Every 500 μ L ddH20.5g dextran sulfates are added in 0, overnight in 37 DEG C of concussions, have waited for
Brief centrifugation mixing after full thawing.
8. a kind of method using few sequence detection forest chromosome end according to claim 1, which is characterized in that institute
The processing method for stating salmon sperm dna is：The salmon sperm dna dissolved at room temperature (10mg/mL) high pressure sterilization is handled into 10min, boiling
Boiling boiling makes its DNA be denaturalized 10min, -20 DEG C of preservations.
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| WO1997024435A1 (en) * | 1995-12-29 | 1997-07-10 | Darwin Molecular Corporation | Genes and gene products related to werner's syndrome |
| CN104073568A (en) * | 2014-07-18 | 2014-10-01 | 西南大学 | Fluorescence in situ hybridization method for metaphase chromosome of mulberry |
| CN106987590A (en) * | 2017-05-25 | 2017-07-28 | 河南省农业科学院 | One cultivates peanut oligonucleotide probe and its design method and application method |
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