CN101173272B - Cotton DNA in situ hybridization technique - Google Patents
Cotton DNA in situ hybridization technique Download PDFInfo
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- CN101173272B CN101173272B CN2007101758134A CN200710175813A CN101173272B CN 101173272 B CN101173272 B CN 101173272B CN 2007101758134 A CN2007101758134 A CN 2007101758134A CN 200710175813 A CN200710175813 A CN 200710175813A CN 101173272 B CN101173272 B CN 101173272B
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Abstract
The invention provides a method of hybridization in situ (Fiber-FISH) of cotton DNA fiber fluorescence. The invention adopts jackknife method to prepare karyon and drainage to pull the DNA fiber and then achieves hybridization in situ of DNA fiber fluorescence by using genome and 45S rDNA as the probes. During extracting the karyon by jackknife method, the phenols combined matter such as PVP40 isadded into the extracting solution and the hurtful medicine such as mercaptoethanol is avoided. The success rate reaches to a hundred percent. During pulling the DNA fiber by drainage, the influence of surface tension is avoided and the accumulation and fracture of the DNA fiber can be avoid, which are caused by uneven load when pushing the glass slide, therefore the prepared DNA fiber is lank, even and integrated. The genome and 45S rDNA label the probes by indentation translation method or random priming to achieve hybridization in situ of DNA fiber fluorescence and the legible and bright signal image can be obtained.
Description
Technical field
The invention belongs to the molecular cytogenetics field, be specifically related to a kind of dna fiber fluorescence in situ hybridization (fiber fluorescent in situ hybridization, Fiber-FISH) technology.
Background technology
DNA fluorescence in situ hybridization (FISH) technology is a kind of important on-radiation hybridization in situ technique that begins to grow up the beginning of the eighties at the end of the seventies.Its ultimate principle is with the nucleic acid molecule mark (as biotin-dUTP or digoxigenin-dUTP) of dna probe with special modification, according to corresponding sequence hybridization on DNA-DNA or DNA-RNA basepairing rule and the karyomit(e).When carrying out in situ hybridization,, make it to take place sex change earlier with high temperature or alkaline purification dna molecular; When temperature descends or the pH value returns to neutrality, the DNA of sex change can form hydrogen bond and return to original duplex structure again according to base complementrity paired principle.If the source difference of strand two, having only base sequence between them is homology complementation or portion homologous when complementary, could all or part of renaturation, produce molecular hybridization.Because probe has fluorescent substance, therefore the site of hybridization can directly be observed under fluorescent microscope.Utilize this technology can detect location, qualitative, the relative quantification branch folding of dna sequence dna on karyomit(e) or dna fiber.
Along with the development of technology and constantly perfect, fluorescence in situ hybridization is used more and more obvious with the superiority of research on plant: (1) is compared with come the hybridization histochemistry of label probe with radioactive substance, it does not need radio isotope, safe and reliable, do not need special methods to handle, detection time is short, and background is clear, detection sensitivity height, but substrate long storage and non-inactivation; (2) compare with other on-radiation hybridization in situ technique, fluorescein-labeled probe of its available difference and same target DNA are hybridized, and promptly carry out multiple labelling, a plurality of dna sequence dnas of one-time positioning; (3) hybridization signal can amplify step by step, carries out quantitatively or the non-quantitation analysis, and can detect and handle the faint signal of hybridization by the computer software image analysis system, makes it visual stronger.
The development of FISH technology is advanced along two lines: be to adopt different probes on the one hand, thereby derive many FISH new technologies, as Multicolor-FISH, CGH, GISH, CISS, BAC-FISH, Chromosome Painting, Reverse ChromosomePainting or the like; Then be the resolving power of making great efforts to improve the FISH technology on the other hand, target is developed into dna fiber from Metaphase Chromosome, make its resolving power develop into 1kb by 1Mb, this has further expanded FISH The Application of Technology field, becomes a representative technology of molecular cytobiology.
The application facet of different probe is that the GISH technology of probe can be located the position of exogenous dna fragment on karyomit(e), size, insertion point etc. with the genome.The chromosome nonhomologous that Durnam (1985) at first is applied to GISH somatic cell hybrid detects.This laboratory adopts the GISH method that tetraploid origin of cotton and evolution are studied.With the multi-color fluorescence in situ hybridization (Multicolor-FISH) of different fluorescein-labelled probes, can locate the distribution of different probe sequence simultaneously.Nineteen ninety Nederlof etc. has created multicolor fluorescence in situ hybridization.They make single, double, three marks with vitamin H, AAF (amino acetylfluorene) and three kinds of haptens of CP to different probe, again with these three kinds of haptens accordingly respectively mark fluorescein isothiocyanate (FITC, green), ammonia first tonka bean camphor acetate (AMCA, blue) and rhodamine (TRITC, redness) antibody detects fluorescence, and this technology can be observed 7 target chromosomes at most simultaneously.Be mixed into probe with normal gene group DNA and aberrant gene group DNA; with normal Metaphase Chromosome is that (comparativegenomic hybridization, CGH) technology can find out that DNA in the genome increases or the zone of disappearance according to the strength difference of two kinds of probe signals for the comparative genome hybridization of target.With single chromosome library is probe, is that chromosome painting (Chromosome Painting) technology of target is particularly suitable for detecting many bodies of karyomit(e) and transposition with the abnormal chromosome.With micro-isolating abnormal chromosome section is probe, is the source that the reverse chromosome painting technology (ReverseChromosome Painting) of target can be determined the abnormal chromosome section with the normal dyeing body.With BAC, YAC, cosmid library is probe, can be simultaneously to a plurality of library clones position, sequencing.Utilization BAC-FISH technology, now with bacterial leaf spot resistant ospc gene Xa21, cotton rRNA gene, blast resisting Pi25 (t), anti-greenery cicada gene Glh and anti-paddy rice verticillium RTSV successfully navigate on the karyomit(e).Directly on target DNA be with the probe primer carry out original position synthetic primer original position DNA synthetic technology (primed in situ DNA synthesis, PRINS) and original position round pcr (PCR in situ) improved the efficient of in situ hybridization and the sensitivity of detection.
The resolving power of FISH technology in order to carry out the dna sequence dna location more accurately, must be made great efforts to improve in the raising aspect of resolving power.Its resolving power is meant the minor increment that two different dna probes can be detected.In the factor of decision FISH resolving power, the most important thing is the degree of enrichment (condensation) of employed target DNA on karyomit(e) or dna fiber, that is the three-dimensional space parcel degree of DNA on chromosomal structure or dna fiber, degree of enrichment is high more, and resolving power is low more.Early stage FISH is many, and it has film-making easy with the carrier of Metaphase Chromosome as target DNA, can stablize the advantage that keeps chromosome structure and form, but its resolving power has only 1～3Mb.In order to obtain the carrier of the low karyomit(e) of degree of enrichment as target DNA, Inazawa etc. (1994), Haaf and Ward (1994) karyomit(e) and with centrifugal machine power extended Metaphase Chromosome mitosis prophase of the employing respectively, resolving power is brought up to about 200kb, but the film-making difficulty improves the limited in one's ability of resolving power.Albini etc. (1992) have successfully carried out the Position Research of two tumor-necrosis factor glycoproteinss on the rye pachytene chromosome.Zhong etc. (1996) adopt the reduction division pachytene chromosome of tomato as target, have carried out the Position Research of dna single one sequence, make level of resolution reach 100kb, but film-making acquire a certain degree of difficulty.In order to break through the restriction of FISH resolving power, must consider for a change chromatinic original space structure of people.Heng etc. (1992) to being in G1 and the interphase nuclei of G2 between the phase handled, discharge free chromatic thread (free chromatin) with alkaline molten born of the same parents' agent (alkaline lysisbuffer), and its FISH level of resolution is near 10kb.Although free chromatic thread has lost original cell spaces structure, it is still DNA and proteinic compound macromole.On the basis of the thinking of (1992) such as Heng, Wiegant (1992) and Windle (1993) further expect with stronger denaturing agent chromatic thread being handled, and allow dna molecular isolate dna fiber (extended DNA fiber) fully from protein.Handle nucleus (Wiegant, 1992), the free single cell culture liquid (Parra and Windle, 1993) of interval or nucleus (zhong, 1996 that propose by the alkaline denaturing agent that contains high salt; Scott, 1998), or separate the dna fiber that macromole DNA prepares with PFGE, its DNA range of extension all can make FISH resolving power reach 1kb even lower.This high-resolution dna fiber fluorescence in situ hybridization (Fiber-FISH) has its original advantage on using, as analyze overlapping sets of cloning (Heiskanen, 1994), detect chromosome rearrangement (Heiskanen, 1995), judge ID and mrna length (Heiskanen, 1995), measure the size (Shiels in length dna site, 1997) and finally quicken map based cloning (Laan, 1996).
Fiber-FISH compares with common FISH has incomparable advantage, and the first, its resolving power improves greatly, and resolving power is 1～2kb, and sensitivity can reach 200bp, and is very nearly the same with the molecular biological restriction map of routine.The second, can directly determine the Rankine-Hugoniot relations between the different dna sequence dnas, have fast advantage directly and accurately.The 3rd, the extension degree that the various different methods that stretch dna fiber obtain can carry out the quantitative analysis of DNA substantially near the theoretical value 2.97kb/ μ m of b form dna molecule in the dna double spin model.For example, measure the size and the copy number of a certain gene or specific dna sequence, gap (gap) size between the contig in the physical mapping (contig) etc.Therefore, genomic Fiber-FISH analyze be called again the mapping of quantity D NA fiber (quantitative DNA fiber mapping, QDFM).The 4th, hybridization signal all shows as discrete beads shape (beads-on-string).
The shortcoming that Fiber-FISH has, the first, the chromosome sectioning difficulty; The second, the requirement height of digitized image analytical system.Arrange because the hybridization signal of Fiber-FISH is the pearl string data, and signal a little less than, require that therefore weak signaling point is had highly sensitive, can the range gate capture point in 0.5～4s, and professional digitized image analytical system that can meticulous measuring probe length.What use always in Fiber-FISH at present is SenSys CCD (chargecoupled device) photographic system (Photometrics), and process software mainly contains IPLab, V++Precision Digital Imaging System etc.The 3rd, can not directly determine chromosomal position and sequence number, need be used in combination with technology such as other FISH.The 4th, dna fiber easy fracture, extension degree differ and are covered the quantitative analysis results error cause by protein.The 5th, the influence that the polymorphism of strand of dna townhouse row causes quantitative analysis need be carried out the comparative analysis of the different hybridization signals of same probe.
Since Henry, Wiegant, people such as Parra and Windle has stretched dna fiber in situ hybridization (Fiber-FISH) technology and has obtained using widely in many aspects such as human genome research, physical map structure, chromosome aberration researchs since carrying out the in situ hybridization achieving success on the dna fiber of preparing (extended DNA fiber).People such as Fransz in 1996 are at first with little tumor-necrosis factor glycoproteins and cosmid linkage group (contig) research of this technology introduced plant Arabidopis thaliana and tomato, and people successfully use in various plants such as paddy rice, corn, wheat, beet and rape afterwards.The Fiber-FISH technology is used very successful in other plants such as paddy rice, Arabidopis thaliana at present, in some cases, the fiber-FISH instrument that almost is absolutely necessary, as when carrying out the assembling of BAC overlapping sets of cloning, because the existence of tumor-necrosis factor glycoproteins, when not having effective BAC library in some zone or not having enough dna markers owing to low recombination fraction, will on physical map, form gap, almost in the report of all overlapping sets of cloning, all there is such gap, utilizes Fiber-FISH can estimate the size of gap apace.Fransz etc. (1996) have carried out the high resolving power physical mapping by Fiber-FISH on Arabidopis thaliana, analyzed the contig that comprises 3 cosmid, cover the long genomic fragment of 89kb.This method is in conjunction with polychrome FISH, and resolving power reaches 0.7kb.
Yet the cotton gene group is big, and cotton tissue is rich in secondary metabolic substds such as gossypol, tannin and terpenes.Up to now, both do not had jackknife to prepare nuclear report, do not prepared the report of dna fiber yet, more do not have the report of dna fiber fluorescence in situ hybridization (Fiber-FISH).
Summary of the invention
The objective of the invention is to provides a kind of dna fiber fluorescence in situ hybridization (Fiber-FISH) method at above-mentioned deficiency.
The present invention at first prepares dna fiber by drainage, and then carries out fluorescence in situ hybridization.
The concrete operations that drainage prepares dna fiber are: the nucleus suspension is dropped in or spreads upon on the slide glass A, examine with nucleus lysate lysing cell, end with another slide glass B touches lysis buffer, and slide glass A one end slowly lifted, while slide glass B hauls liquid and smears downwards, again through fixing, the dry dna fiber that gets.Certainly, preferably can be with of the end line of nucleus suspension at slide glass A, after the lysate cracking, touch lysis buffer with the end of another slide glass B and will sprawl side, and slide glass A is coated with a nucleolate end slowly lifts, while slide glass B hauls liquid and smears downwards, again through fixing, dry.And then carry out fluorescence in situ hybridization with designed probe.The purpose of drainage is for the dna fiber that stretches, this method slice-making quality height, and dna fiber is evenly distributed, and is difficult for producing fracture or losing.
The nucleus suspension preferably prepares by jackknife, concrete grammar is to cut biomaterial with the freezing sharp cutter of sterilization in the nucleus extracting solution, makes it into the homogenate shape, with after filter the of short duration centrifugal nucleus that obtains in back step by step, resuspended with the nucleus extracting solution, making its final concentration is 5 * 10
6About nucleus/mL.
The above-mentioned employed filter membrane aperture of filtering step by step should be selected according to the nuclear size of object, and for cotton yellow cotyledon, can select the aperture is that the filter membrane that is respectively 100 μ m, 50 μ m, 30 μ m filters step by step.
The preferred scheme of the present invention is to adopt jackknife to prepare nucleus, drainage prepares dna fiber, make probe with vitamin H or digoxin to incise translation method or random priming marker gene group DNA and/or 45S rDNA, cotton DNA fibre is carried out fluorescence in situ hybridization, take pictures, handle and process with the Zeiss fluorescent microscope.
It is slow and steady that drainage stretching dna fiber, speed are wanted.Slide glass A should guarantee that when lifting the nucleus suspension does not flow downward, thereby, the suitably dry back of nucleus suspension (as air drying 3～5 minutes) can be dripped the cracking of nucleus lysate again, and, when slide glass A one end lifts, touch the last lysis buffer of slide glass A with slide glass B and will sprawl side, surface tension by liquid can be so that suspension flow downward, pass through the traction of slide glass B then, make liquid walk equably downwards, the DNA that is free in the liquid just can form parallel dna fiber.The angle that slide glass A lifts is advisable with (30～60 ° of scopes), is preferably 45 °.
With genomic dna and/or 45S rDNA, with nick-translation mark biotinylated probes, perhaps with random priming mark digoxigenin-probe.Probe behind the mark carries out sex change, hybridizes with the ready slide glass that contains target DNA of the certain processing of process, and the wash-out detection of hybridization back, acquired information is handled and is processed, and draws the analysis conclusion.
For the preparation plant nucleolus, prepare nucleus by jackknife and will help to improve the nuclear success ratio of preparation.
Drainage stretching dna fiber can make dna fiber even and straight, helps the carrying out and the interpretation of result of follow-up test.
Vitamin H or digoxin are carried out fluorescence in situ hybridization to incise translation method or random priming marker gene group or 45SrDNA probe, can obtain the hybridization signal of chaining pearl, take a picture with the Zeiss fluorescent microscope and handle and processing.
The inventive method is specially adapted to the cotton DNA fibre fluorescence in situ hybridization, during preparation cotton cells nuclear, the optimal seeking of raw and processed materials is the light yellow beggar's leaf of cotton children, the preparation method of nucleus suspension is: get cotton yellow cotyledon, add and contain 2% PVP40 nucleus extracting solution, cotton yellow cotyledon is cut into the homogenate shape, classified filtering, the centrifugal supernatant liquor of removing, precipitation is resuspended with the nucleus extracting solution, promptly gets nucleus suspension.Operation at low temperatures as far as possible in the above-mentioned preparation process.Prepare dna fiber with drainage again after preparing the nucleus suspension.
The prepared dna fiber of the inventive method compared with prior art, adopt jackknife to prepare nucleus success ratio 100%, and preparing nucleus, polishing has only about 30%, and the easy purifying of nucleus of jackknife preparation, without toxic reagents such as mercaptoethanols, and simple and quick, operate at every turn and can finish in 30 minutes; Drainage prepares dna fiber, and is parallel and scattered, success ratio 100%; Dna fiber fluorescence in situ hybridization signal is clear obviously, the success ratio height, can be used to carry out the chromatinic evaluation of physical positioning, allos, translocator and the aneuploid of the evaluation of height or moderate multiple dna sequence dna, low copy or single-copy DNA sequence and genetic marker evaluation, genomic dna-FISH research, BAC-FISH location, the structure of physical chromosomal map spectrum, sibship is determined and transgenosis detection etc.
Description of drawings
Fig. 1 is the cotton cells nuclear photo that extracts;
Fig. 2 is a drainage tensile cotton DNA fibre photo;
Fig. 3 is to be dna probe fiber fluorescence in situ hybridization photo with the genome;
Fig. 4 is to be dna probe fiber fluorescence in situ hybridization photo with 45S rDNA.
Embodiment
Further set forth the present invention below in conjunction with specific embodiment.Should be appreciated that these embodiment only are used to illustrate the present invention, and can not limit protection scope of the present invention.
Embodiment 1 is the cotton DNA fibre fluorescence in situ hybridization technique of probe with the genome:
Material: inferior No. 1 of Asiatic cotton kind stone system (cotton research science and trade company of the Chinese Academy of Agricultural Sciences)
1, the preparation of yellow cotyledon
Fine sand dries in the air agglomerating to holding after the poach sterilization, the degree of loosing one's grip and promptly loosing.Get inferior No. 1 cotton seeds of stone system, cut off the most advanced and sophisticated skin of planting, put into about 40 ℃ warm water and soaked seed 12～24 hours, then, be seeded in the fine sand that scalding is crossed, cultivate in 37 ℃ of constant temperature darkrooms, and the cotyledon that promptly grows young light yellowization after 2～3 days comes.
2, nuclear extraction
(1) gets the young light yellow beggar's leaf of 2 grams, put into the culture dish of the diameter 5cm that places on ice immediately.
(2) the nucleus extracting solution (MgSO of adding 12.5ml in the culture dish
410mmol/L, KCl 5mmol/L, HEPES 0.5 mmol/L, DTT 1mg/ml, Triton X-1000.25%, PVP40 2%)
(3) with the aseptic sharp cutter of sterilization blade being cut into the homogenate shape, is the nylon membrane filtration of 100 μ m, 50 μ m and 30 μ m by the aperture respectively with homogenate, and all operations needs finish on ice;
Centrifugal under (4) 4 ℃ (16000 * g) 40 seconds, remove supernatant liquor, the nucleus that throw out is suspended in 125 μ L extracts in the damping fluid;
(5) get 1 μ L nucleus suspension, with DAPI dyeing, check nuclear concentration under fluorescent microscope, through counting, nuclei concentration is 5 * 10
6Individual nucleus/mL.The nucleus suspension of preparation as shown in Figure 1.
3, the preparation of dna fiber
(1) gets 1 μ L nucleus suspension,, come and go and smear being aligned at the end of dustless slide glass A.Dry air 4 minutes.
(2) on the nucleus that coats, add 30 μ L karyolysis damping fluids (0.5%SDS, 5mmol/L EDTA, 100mmol/L Tris, pH7.0), incubation 9min under the room temperature.
(3) get another clean slide glass B, one end edge gently exposing cell karyolysis damping fluid will sprawl side, the A slide glass is coated with a nucleolate end to be lifted, slowly make it horizontal by miter angle, slide glass B hauls liquid and smears whole slide glass A simultaneously, and the free dna molecular just can form parallel dna fiber.
(4) behind the dry air 10min, fixed dna fiber 2min in Carnoy fluid, 60 ℃ of oven dry 30min.
(5) sampling sheet DAPI dyeing, the quality of inspection dna fiber under fluorescent microscope.Film-making can be in-20 ℃ of following kept dry numbers (6～10) moon.The dna fiber of preparation as shown in Figure 2.
4, the preparation of probe
Random priming: digoxin (Digoxigenin-dUTP) is marked in the ice bath to be prepared
Preparation dNTP mixed solution: volume 10 μ L
Get 0.4mM dATP 2.5 μ L
0.4mM?dGTP 2.5μL
0.4mM?dCTP 2.5μL
0.4mM?dTTP 0.6μL
0.4mM?DiG?dUTP 0.9μL
Add 10 * buffer, 2 μ L successively again
The template DNA that interrupts (2 μ g/ μ l) 2 μ L
Add deionized aqua sterilisa 4 μ L
Add Mix Enzyme 2 μ L behind the mixing again
Markers step:
1. put into 15 ℃ of 90min
2. 65 ℃ of 10min deactivations
3. add the NaAc (pH5.2) of 1/10 volume, and then add 3 times of dehydrated alcohols, precipitation
4.-20 a ℃ precipitation is spent the night
5. second day 4 ℃ 12000 leave heart 30min
6. precipitation is washed (will note tube wall) slightly with 70% ethanol, dries
7. 65 ℃ are dissolved among the 20 μ LTE, be distributed into be stored in 10 tubules-20 ℃ standby
5, the preparation of film-making
(1) film-making dried overnight in 60 ℃ of baking ovens;
(2) with the RnaseA (2 * SSC preparation) of 100 μ g/ml, 37 ℃ of 30min;
(3) 2 * SSC float cover plate, 2 * SSC, 3 * 3min washing;
(4) 0.1%～0.01%Pepsin (10mM HCl preparation), 37 ℃ of 20min;
(5) 2 * SSC float cover plate, 2 * SSC, 2 * 3min washing (or 1 * PBS 2 * 3 minutes);
(6) 4% Paraformaldehyde 96s (W/V, matching while using) are in 37 ℃ of fixing 5min, 2 * SSC, 2 * 3min washing;
(7) 70%-90%-100% ethanol dehydration, every grade of 3min dries standby.
(if the stretching dna fiber is very clean, 2. to 7. the step can economize)
6, the preparation of hybridization mixed solution
(1) the hybridization solution cumulative volume is 20 μ L:
100% deionized formamide, 10 μ L
50% T 500,4 μ L (it is standby to put into 70 ℃ of dissolvings)
20 * SSC (standard sodium citrate salt), 2 μ L
10%SDS (sodium laurylsulfonate) 1 μ L (it is standby to put into 70 ℃ of dissolvings)
Fluorescein-labeled probe 1 μ L
Supply (the available DNA of blockading) with the deionized water of sterilization during less than 20 μ L
(2),, change over to and handle 10～15 minutes in the frozen water in 97 ℃ of sex change 5 minutes with the hybridization solution mixing.
7, hybridization
(1) the hybridization drop of getting 20 μ L sex change adds the plastic film cover plate in film-making, and the packing back is in 80 ℃ of co-variation 5min;
(2) change over to hybridize in 37 ℃ of water-baths and spend the night, or 2～3 days.
8, wash-out and signal detection
(1) 2 * SSC washes, 5 minutes;
(2) 2 * SSC wash, and 42 ℃, 10 minutes;
(3) 2 * SSC wash, 5 minutes;
(4) 1 * 4T wash, 5 minutes;
(5) 4M, 37 ℃ of incubations, 30 minutes;
(6) 1 * 4T wash, 2 minutes;
(7) dry (film-making can not be seen dried), add antibody A nti-DIG-Rhodamine, add-on is 30 μ L/ sheets, with cover plate, puts into 37 ℃ of water-baths 1 hour;
(8) 1 * TNT wash, and 3 minutes, 2 * SSC washed, 3 minutes;
(9) drying (film-making can not be seen dried) dyed 10 minutes with the DAPI lining.
(10), 2 * 3 minutes, dry back with anti-fluorescence decay agent mounting with 1 * PBS washing.
Zeiss fluorescent microscope detected result is amplified 1000 times as shown in Figure 3, and hybridization signal is discrete chain pearl as seen from the figure, under 1000 times clearly.
Embodiment 2 is the cotton DNA fibre fluorescence in situ hybridization technique of probe with 45S rDNA:
Material: inferior No. 1 of Asiatic cotton kind stone system (cotton research science and trade company of the Chinese Academy of Agricultural Sciences)
1, the preparation of yellow cotyledon
Fine sand dries in the air agglomerating to holding after the poach sterilization, the degree of loosing one's grip and promptly loosing.Get inferior No. 1 cotton seeds of stone system, cut off the most advanced and sophisticated skin of planting, put into about 40 ℃ warm water and soaked seed 12～24 hours, then, be seeded in the fine sand that scalding is crossed, cultivate in 37 ℃ of constant temperature darkrooms, and the cotyledon that promptly grows young light yellowization after 2～3 days comes.
2, nuclear extraction
(1) gets the young light yellow beggar's leaf of 2 grams, put into the culture dish of the diameter 5cm that places on ice immediately.
(2) the nucleus extracting solution (MgSO of adding 12.5ml in the culture dish
410mmol/L, KCl 5mmol/L, HEPES 0.5mmol/L, DTT 1mg/ml, Triton X-1000.25%, PVP40 2%)
(3) with the aseptic sharp cutter of sterilization blade being cut into the homogenate shape, is the nylon membrane filtration of 100 μ m, 50 μ m and 30 μ m by the aperture respectively with homogenate, and all operations needs finish on ice;
Centrifugal under (4) 4 ℃ (16000 * g) 40 seconds, remove supernatant liquor, the nucleus that throw out is suspended in 125 μ L extracts in the damping fluid;
(5) get 1 μ L nucleus suspension, with DAPI dyeing, check nuclear concentration under fluorescent microscope, through counting, nuclei concentration is 5 * 10
6Individual nucleus/mL.
3, the preparation of dna fiber
(1) gets 1 μ L nucleus suspension,, come and go and smear being aligned at the end of dustless slide glass A.Dry air 4 minutes.
(2) on the nucleus that coats, add 30 μ L karyolysis damping fluids (0.5%SDS, 5mmol/L EDTA, 100mmol/L Tris, pH7.0), incubation 9min under the room temperature.
(3) get another clean slide glass B, one end edge gently exposing cell karyolysis damping fluid will sprawl side, the A slide glass is coated with a nucleolate end to be lifted, slowly make it horizontal by miter angle, slide glass B hauls liquid and smears whole slide glass A simultaneously, and the free dna molecular just can form parallel dna fiber.
(4) behind the dry air 10min, fixed dna fiber 2min in Carnoy fluid, 60 ℃ of oven dry 30min.
(5) sampling sheet DAPI dyeing, the quality of inspection dna fiber under fluorescent microscope.Film-making can be in-20 ℃ of following kept dry numbers (6～10) moon.
4, the preparation of probe
Nick-translation: vitamin H (Biotin-dUTP) carries out mark
(1) gets template DNA 2 μ L (final concentration 1ug/ μ L), fragment length 500～600bp
(2) add two pure water 14 μ L
(3) add (Biotin-Nick Translation Mix) 4 μ L
(4) put into 15 ℃ of 90min of PCR instrument
(5) add 0.5M EDTA 1 μ L
(6) put into 65 ℃ of 10min of PCR instrument
(7)-20 ℃ preservation is standby
5, the preparation of film-making
(1) film-making dried overnight in 60 ℃ of baking ovens;
(2) RnaseA (2 * SSC preparation) of usefulness 100ug/ml, 37 ℃ of 30min;
() 2 * SSC floats cover plate, 2 * SSC, 3 * 3min washing;
(4) 0.1%～0.01%Pepsin (10mM HCl preparation), 37 ℃ of 20min;
(5) 2 * SSC float cover plate, 2 * SSC, 2 * 3min washing (or 1 * PBS 2 * 3 minutes);
(6) 4% Paraformaldehyde 96s (W/V, matching while using) are in 37 ℃ of fixing 5min, 2 * SSC, 2 * 3min washing;
(7) 70%-90%-100% ethanol dehydration, every grade of 3min dries standby.
(the dna fiber quality is fine, 2. to 7. the step can economize)
6, the preparation of hybridization mixed solution
(1) the hybridization solution cumulative volume is 20 μ L:
100% deionized formamide, 10 μ L;
50% T 500,4 μ L (it is standby to put into 70 ℃ of dissolvings);
20 * SSC (standard sodium citrate salt), 2 μ L;
10%SDS (sodium laurylsulfonate) 1 μ L (it is standby to put into 70 ℃ of dissolvings);
Fluorescein-labeled probe 1 μ L;
Supply (the available DNA of blockading) with the deionized water of sterilization during less than 20 μ L;
(2),, change over to and handle 10～15 minutes in the frozen water in 97 ℃ of sex change 5 minutes with the hybridization solution mixing.
7, hybridization
(1) the hybridization drop of getting 20 μ L sex change adds the plastic film cover plate in film-making, and the packing back is in 80 ℃ of co-variation 5min;
(2) change over to hybridize in 37 ℃ of water-baths and spend the night, or 2～3 days.
8, wash-out and signal detection
(1) 2 * SSC washes, 5 minutes;
(2) 2 * SSC wash, and 42 ℃, 10 minutes;
(3) 2 * SSC wash, 5 minutes;
(4) 1 * 4T wash, 5 minutes;
(5) 4M, 37 ℃ of incubations, 30 minutes;
(6) 1 * 4T wash, 2 minutes;
(7) dry (film-making can not be seen dried), add antibody FITC-Avidin, add-on is 30 μ L/ sheets, with cover plate, puts into 37 ℃ of water-baths 1 hour;
(8) 1 * TNT wash, and 3 minutes, 2 * SSC washed, 3 minutes;
(9) dry (film-making can not be seen dried), add antibody Biotin anti-Avidin, add-on is 30 μ L/ sheets, with cover plate, puts into 37 ℃ of water-baths 1 hour;
(10) 1 * TNT wash, and 3 minutes, 2 * SSC washed, 3 minutes;
(11) dry (film-making can not be seen dried), add antibody FITC-Avidin, add-on is 30 μ L/ sheets, with cover plate, puts into 37 ℃ of water-baths 1 hour;
(12) drying (film-making can not be seen dried) dyed 10 minutes with the DAPI lining.
(13), 2 * 3 minutes, dry back with anti-fluorescence decay agent mounting with 1 * PBS washing.
Zeiss fluorescent microscope detected result as shown in Figure 4, as seen from the figure, hybridization signal is discrete chain pearl, under 1000 times clearly.
Claims (10)
1. dna fiber fluorescence in-situ hybridization method, it comprises step:
1) the nucleus suspension is dropped in or spreads upon on the slide glass A, examine with nucleus lysate lysing cell, end with another slide glass B touches lysis buffer, and slide glass A one end slowly lifted, and guarantee that the nucleus suspension does not flow downward, while slide glass B hauls liquid and smears downwards, again through fixing, the dry dna fiber that gets;
2) carry out fluorescence in situ hybridization with designed probe.
2. the method for claim 1 is characterized in that, also comprises the fluorescence in situ hybridization result is detected.
3. the method for claim 1 is characterized in that, the biomaterial of preparation dna fiber is a plant, and adopts jackknife to prepare the nucleus suspension.
4. method as claimed in claim 3 is characterized in that, described biomaterial is a cotton yellow cotyledon.
5. method as claimed in claim 4, it is characterized in that, the method for preparing the nucleus suspension is: with the freezing sharp cutter of process sterilization, cotton yellow cotyledon is cut into the homogenate shape in being added with the nucleus extracting solution, the of short duration centrifugal supernatant that goes after filtering step by step, precipitation is resuspended with the nucleus extracting solution.
6. method as claimed in claim 5 is characterized in that, the filter membrane that adopts the aperture to be respectively 100 μ m, 50 μ m, 30 μ m filters step by step.
7. method as claimed in claim 5 is characterized in that, contains 2% PVP40 in the described cell extract.
8. as each described method of claim 1～6, it is characterized in that the concentration of described nucleus suspension is 5 * 10
6Nucleus/mL.
9. as each described method of claim 1～6, it is characterized in that the angle that slide glass A one end lifts is 30～60 °.
10. as each described method of claim 1～6, it is characterized in that with genomic dna of the same race or 45S rDNA obtaining the result and collecting, handle and processing with random priming or incise translation method label probe and carry out the dna fiber fluorescence in situ hybridization through the Zeiss fluorescent microscope.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| BRPI0917094B1 (en) * | 2008-08-08 | 2018-01-16 | Bayer Cropscience Nv | Methods for identification of processed plant fiber, genome analysis of a fiber producing cotton plant, and isolation of naturally occurring DNA from processed cotton plant fibers, use of said methods, as well as methods for isolation of a naturally occurring DNA from knitted fabric or cloth, to determine the relative amounts of different cotton plant fibers in a blend of processed cotton fibers, and to certify the identity of marketed cotton fibers |
| CN101575597B (en) * | 2009-05-21 | 2011-10-05 | 中国农业大学 | Kit for quickly extracting plant genome and applications thereof |
| CN102994614B (en) * | 2011-09-09 | 2014-04-09 | 中国科学院生态环境研究中心 | Fluorescent in-situ hybridization method for detecting plant rhizosphere microbe |
| CN104313164B (en) * | 2014-10-30 | 2016-05-04 | 中国农业科学院棉花研究所 | Differentiate a complete set of chromosomal method of cotton D genome and D sub-genome |
| CN108589287A (en) * | 2018-04-03 | 2018-09-28 | 四川大学 | A kind of in-situ preparation method of fluorescence cotton fiber |
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| CN101148663A (en) * | 2007-08-30 | 2008-03-26 | 中国农业科学院棉花研究所 | Preparation for cotton DNA fibre by cutting and drainage method |
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| CN101148663A (en) * | 2007-08-30 | 2008-03-26 | 中国农业科学院棉花研究所 | Preparation for cotton DNA fibre by cutting and drainage method |
Non-Patent Citations (6)
| Title |
|---|
| Lijia Li等.A Novel Approach to Prepare Extended DNA Fibers in Plants..Cytometry Part A63 2.2005,63(2),摘要、正文115页左栏第4-7段. |
| Lijia Li等.A Novel Approach to Prepare Extended DNA Fibers in Plants..Cytometry Part A63 2.2005,63(2),摘要、正文115页左栏第4-7段. * |
| P.F.Fransz等.High-resolution physical mapping in Arabidopsis thaliana andtomato by fluorescence in situ hybridization to extended DNAfibres..The Plant Journal9 3.1996,9(3),正文428页右栏第3-4段. |
| P.F.Fransz等.High-resolution physical mapping in Arabidopsis thaliana andtomato by fluorescence in situ hybridization to extended DNAfibres..The Plant Journal9 3.1996,9(3),正文428页右栏第3-4段. * |
| 殷剑美，等.基于棉花BAC文库的大片段核DNA的提取方法。.《分子植物育种》2 3.2004,2(3),正文355页右栏第1段. |
| 殷剑美等.基于棉花BAC文库的大片段核DNA的提取方法.《分子植物育种》2 3.2004,2(3),正文355页右栏第1段. * |
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