CN104120171A - Radiation induced cell chromosome morphology detection method - Google Patents
Radiation induced cell chromosome morphology detection method Download PDFInfo
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Abstract
The invention relates to a radiation induced cell chromosome morphology detection method. The method comprises the following steps: 1, preparing a fluorophor directly labeled fluorescent centromere DNA probe; 2, carrying out fluorescence in-situ hybridization on the fluorescent DNA probe obtained in step 1 and a radiation induced cell chromosome specimen; and 3, detecting the centromere and chromosome morphology under a fluorescence microscope. The method adopts the directly labeled fluorescent centromere DNA probe to rapidly analyze the radiation induced cell chromosome morphology through a fluorescent in-situ hybridization (FISH) technology; and compared with common indirectly labeled probes, the directly labeled probe simplifies the FISH operation step, and enables the detection signal to be very good under the fluorescence microscope after the hybridization at 37DEG C 5-12h and simply washing.
Description
Technical field
The invention belongs to molecular cytogenetics field, be specifically related to prepare method and the application in biomedicine, radiation biology field of the general centromeric probe of direct mark.
Background technology
Chromosome specimen fluorescence in situ hybridization (fluorescence in situ hybridization, FISH) technology is applied comparatively extensive in radiation biology field, this technology has advantages of visual and clear image, rapidly and efficiently analyzes, somatoblast specification of quality is so not tight, to microscopy, analyzer is less demanding, especially judge that translocation is quicker than conventional G Banded method, accurately, thereby solved the difficult problem that dosage estimation need to be counted a large amount of cells.At present, FISH technology is mainly used in retrospective dose reconstruction, to being previously subject to ionization radiation irradiation personnel and chronic being subject to, according to personnel, carry out dosage estimation (International Atomic Energy Agency.Cytogenetic analysis for radiation dose assessment.A manual technical report series No.405.Vienna:IAEA, 2001:109-115).
α-satellite DNA (alpha satellite DNA) is a unique centromere DNA (centromere DNA who is present in everyone region, chromosomoid kinetochore, CEN-DNA) family, monomer by 171bp is the height series connection repeated fragment that unit forms, repeat hundreds of times to thousands of times, leap reaches the centromere DNA region of 100kb, most of sequence high conservative wherein, but on every karyomit(e), its conserved sequence still has certain otherness (Willard HF, Chromosome-specific organization of human alpha satellite DNA.Am J Hum Genet.1985, 37 (3): 524-532).In FISH technology, general centromeric probe can be combined closely with target DNA; hybridization signal is also stronger; be easy to detect; mainly for detection of chromosomal number, analyze two kinetochore body and centric rings and apply general centromeric probe and accurately distinguish two kinetochore bodies and transposition in conjunction with whole chromosome specific probe.
Desirable probe mark thing, should possess following some: 1. high sensitivity; 2. the combination of marker and nucleic acid probe molecules, can not affect its base pairing specificity; 3. do not affect the main physicochemical property of probe molecule; 4. when adopting enzymatic method to carry out mark, reply enzymic activity has no significant effect; 5. high degree of specificity (Lu Shengdong chief editor. modern molecular biology experimental technique [M]. Beijing: Higher Education Publishing House, 1993.159).
While applying the rear cell dyeing volume morphing of general centromeric probe analyzing radiation induction in existing FISH technology, conventional hapten molecule (vitamin H or digoxin) indirect labelling probe, therefore probe carries out signal iodine and repeatedly develops a film from needing high degree of specificity and high-affinity antibody with the different fluorescent substances of coupling after Chromosomal in situ hybridization, has the problems such as length consuming time, complex operation step, efficiency are low.
Summary of the invention
The object of the invention is to set up a kind of detection method of radiation-induced rear cell dyeing volume morphing, to solve, in prior art, apply the general centromeric probe of indirect labelling and analyze the problems such as chromosome morphology length consuming time, complex operation step.
Object of the present invention can be by realizing by the following technical solutions:
A detection method for radiation-induced rear cell dyeing volume morphing, comprises the steps:
(1), prepare the general centric fluorescent DNA probe of the direct mark of fluorophor;
(2) the fluorescent DNA probe, step (1) being obtained and radiation-induced rear cell chromosome sample fluorescence in situ hybridization;
(3), under fluorescent microscope, detect kinetochore and chromosome morphology.
Wherein, the fluorophor described in step (1) is selected from any one in Cy3, Cy5 or Fluorescein.
Wherein, the DNA sequence dna of the fluorescent DNA probe described in step (1) is from the α-satellite DNA in region, human chromosomal kinetochore.
Preferably, step (1) comprises following two steps:
1) take Healthy People genomic dna as template, pcr amplification people α-satellite DNA fragment;
2) take step 1) α-satellite DNA fragment PCR products of obtaining is template, adds the dUTP with fluorophor in pcr amplification solution, obtains the general centric fluorescent DNA probe of the direct mark of fluorophor.
Wherein, described step (2) specifically comprises cell chromosome film-making, aging, sex change, hybridizes, develops a film, redyes and the step of microscopy.
Preferably, described in step (2), hybridize and be specially: fluorescent DNA probe associating whole chromosome probe prepared by step (1) and radiation-induced rear cell chromosome sample hybridization.Wherein, whole chromosome probe is selected from any in people's whole chromosome probe.
The detection method of the radiation-induced rear cell dyeing volume morphing that the present invention sets up, its advantage is to prepare fast the general centric fluorescent DNA probe of direct mark by conventional PCR method, after fluorescent DNA probe and radiation-induced rear cell chromosome sample fluorescence in situ hybridization, through simple washing, can under fluorescent microscope, detect kinetochore and chromosomal form.The general centric fluorescent DNA probe of direct mark that the present invention uses is compared with indirect labelling probe, without repeatedly developing a film after hybridization and the step such as antibody signal amplification, only need through simple washing, extraordinary signal can under fluorescent microscope, be detected after hybridization.The method is easy, quick, reproducible, and efficiency is high, specificity good.
Accompanying drawing explanation
The behave PCR product agarose gel electrophoresis figure of α-satellite DNA fragment of Fig. 1.M is DNA marker, and 1,2,3 is PCR product parallel samples.
Fig. 2 is the PCR product agarose gel electrophoresis figure of fluorophor mark people α-satellite DNA fragment.M is DNA marker, and Label is the people's α-satellite DNA fragment PCR products with fluorophor mark.
Fig. 3 is the normal lymphocytic cell division Metaphase Chromosome of general centromere DNA probe in detecting with Cy3 mark.
Fig. 4 is the normal lymphocytic cell division Metaphase Chromosome of general centromere DNA probe in detecting with Fluorescein mark.
Fig. 5 is the general centromere DNA probe in detecting 4Gy of Cy3 mark
60chromosomal pair of kinetochore body of metaphase in cell division after Co gammairradiation.
Fig. 6 is the general centromere DNA probe in detecting 12Gy of Cy3 mark
60two kinetochores body and centric ring after Co gammairradiation in premature chromosome condensation.Arrow 1 is depicted as two kinetochores body, and arrow 2 and 3 is depicted as centric ring.
Fig. 7 is No. 2 whole chromosome probe differential staining bodies of the general centromere DNA probe associating people transposition of Fluorescein mark.Arrow 1 and 2 represents No. 2 whole chromosomes of people, and wherein one No. 2 chromosomal long-armed (arrow 2), with No. 10 karyomit(e)s long-armed (arrow 3), transposition occurs; Arrow 4 represents with fluorescently-labeled kinetochore.
Embodiment
Describe by the following examples the detection method of radiation-induced rear cell dyeing volume morphing provided by the invention in detail.Embodiment is only for explaining and explanation content of the present invention below; and should be as limiting the scope of the present invention; biomaterial related in embodiment all can be buied in market; the not special part of describing of related biotechnology all adopts cytogenetics routine techniques, as the routine techniques method providing in < < molecular cloning experiment guide > >.
Embodiment 1: the directly preparation of the general centric fluorescent DNA probe of mark
1, design of primers and synthetic
According to human chromosome centric region α-satellite DNA conserved sequence, carry out degenerated primer design.Upstream primer (Primer-F) SEQ ID NO:1 is: 5 '-GAAGCTTAWSTMACAGAGTTKAA-3 '; Downstream primer (Primer-R) SEQ ID NO:2 is: 5 '-GCTGCAGATCMCMAAGHAGTTTC-3 '; Synthetic by Shanghai Sheng Gong biotechnology limited-liability company.
2, DOP-PCR amplification people α-satellite DNA fragment
1), PCR reaction system is as shown in table 1.
Table 1PCR reaction system
2), PCR reaction conditions: 94 ℃ of sex change 2 minutes (min), 94 ℃ of sex change 40 seconds (s), 50 ℃ of annealing 1min, 72 ℃ are extended 2min, 30 circulations, extend 10min again after loop ends, be cooled to 4 ℃.
Get 10 μ l people α-satellite DNA fragment PCR products, 2% agarose gel electrophoresis, the EB multi-ribbons (referring to accompanying drawing 1) such as visible 171bp, 342bp that dye.
3, direct fluorescence labeling probe
α-satellite DNA the fragment PCR products of above-mentioned acquisition of take is template, adds Cy3-dUTP, Cy5-dUTP or Fluorescein-12-dUTP with fluorophor in pcr amplification solution, obtains the general centric fluorescent DNA probe of direct mark.Step is as follows:
1), PCR reaction system is as shown in table 2.
Table 2PCR reaction system
2), PCR reaction conditions: 94 ℃ of sex change 2min, 94 ℃ of sex change 40s, 50 ℃ of annealing 1min, 72 ℃ are extended 2min, 30 circulations, extend 10min again after loop ends, be cooled to 4 ℃.
Get PCR product after 5 μ l marks, there is an obvious band at 2% agarose gel electrophoresis, the EB visible 171bp place that dyes, and band top is vaporific, show tags success; According to DNA molecular amount Marker, can judge to PCR production concentration (referring to accompanying drawing 2).
4, the purifying of label probe
In PCR product after mark, add the 3M sodium-acetate (pH5.2) of 1/10 volume, and then add the dehydrated alcohol of the precooling of 2.5 times of volumes ,-20 ℃ are spent the night; 4 ℃, 14000rpm, centrifugal 25min, abandons supernatant; With 70% ethanol, clean once again, 4 ℃, 14000rpm, centrifugal 25min, exhaustion supernatant; In precipitation, add appropriate TE Buffer dissolving DNA probe; Prepare the general centric fluorescent DNA probe mixed solution of direct mark (in every 10 μ l containing 5ng fluorescent DNA probe, 55% deionized formamide, 10% T 500,2 * SSC, PH7.0), mix, packing ,-20 ℃, lucifuge is stored.
5, the directly checking of the general centric fluorescent DNA probe of mark: end user's proper splitting lymphocyte in mid-term suspension steam droplets sheet, every chromosomal centric region is visible stronger fluorescent signal all, the general kinetochore of Cy3 mark shows danger signal, and the kinetochore of Fluorescein mark shows green (referring to accompanying drawing 3 and accompanying drawing 4).
Embodiment 2: the directly application of the general centric fluorescent DNA probe of mark
(1) cell cultures: take the about 5ml of volunteer's venous blood, anticoagulant heparin mixes, room temperature
60co gammairradiation is repaired after 2 hours (h), 500 μ l whole bloods are added to (containing 20% foetal calf serum, 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates, 0.2mg/mlPHA) in 4ml RPMI1640 substratum, fully shake up, put 37 ℃ and cultivate 48h harvested cell (with blood donor's age growth proper extension incubation time); Cultivation finishes front 6h, to the colchicine that adds 70 μ 110 μ g/ml in culturing bottle, after mixing, is placed in 37 ℃ of constant incubators and continues to cultivate 6h harvested cells; Wherein, premature chromosome condensation (premature chromosome condensation, PCC) difference of cultural method is after 37 ℃ of constant temperature culture 46h, add Calyculin A (Calyculin A, CA) making its final concentration is 50nmol/L, harvested cell after continuation cultivation 2h;
(2) the hypotonic processing of cell: after cell co-cultivation, the centrifugal 10min of 1000rpm, abandons supernatant, is used the hypotonic 20min of 0.075mol/L KCl5ml;
(3) cell is fixed: in the hypotonic medium that contains hypotonic processing cell, add 1ml stationary liquid to pre-fix, the centrifugal 10min of 1000rpm immediately after mixing, abandons supernatant, then adds 8ml stationary liquid to fix 2 times, the centrifugal 10min of 1000rpm after each fixedly 15min, abandons supernatant; Finally cell is suspended from 1ml stationary liquid; Wherein stationary liquid is that methyl alcohol and Glacial acetic acid are preparation in 3: 1 by volume;
(4) film-making: apply fresh cell suspension and carry out steam droplets sheet, make karyomit(e) be dispersed, mark hybridization region;
(5) aging: in 50 ℃ of constant temperature roasters, 30min;
(6) sex change: add the general centric fluorescent DNA probe mixed solution of the direct mark of 10 μ l above the hybridization region having marked, covered, closes by mounting rubber seal, is placed on roasting sheet machine 73 ℃ of sex change 3min;
(7) hybridization: slide is put into wet box, 37 ℃, spend the night;
(8) develop a film: at 72 ℃, use 0.4 * SSC, 5min develops a film; Then room temperature is used 2 * SSC (0.05%Tween-20), and 30s develops a film; DDW rinses, seasoning;
(9) redye: add 10 μ l DAPI, covered, lucifuge effect 10min;
(10) microscopy: fluorescence microscopy Microscopic observation karyomit(e) is blue, and the kinetochore of Cy3 mark takes on a red color; Visible two signals of two kinetochores body (referring to accompanying drawing 5), centric ring is a visible signal (referring to accompanying drawing 6) only in ring dyeing.
Embodiment 3: the directly general centric fluorescent DNA probe associating whole chromosome probe differential staining body of mark transposition
(1) cell cultures: take the about 5ml of volunteer's venous blood, anticoagulant heparin mixes, add (containing 20% foetal calf serum, 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates, 0.2mg/ml PHA) in 4ml RPMI1640 substratum fully to shake up 800 μ l whole bloods, put 37 ℃ and cultivate 48 harvested cells (with blood donor's age growth proper extension incubation time); Cultivation finishes front 6h, adds the colchicine of 70 μ l10 μ g/ml, after mixing, is placed in 37 ℃ of constant incubators and continues to cultivate 6h;
(2) the hypotonic processing of cell: after cell co-cultivation, the centrifugal 10min of 1000rpm, abandons supernatant, is used the hypotonic 20min of 0.075mol/L KCl5ml;
(3) cell is fixed: in the hypotonic medium that contains hypotonic processing cell, add 1ml stationary liquid to pre-fix, the centrifugal 10min of 1000rpm immediately after mixing, abandons supernatant, then adds 8ml stationary liquid to fix 2 times, the centrifugal 10min of 1000rpm after each fixedly 15min, abandons supernatant; Finally cell is suspended from 1ml stationary liquid; Wherein stationary liquid is that methyl alcohol and Glacial acetic acid are preparation in 3: 1 by volume;
(4) film-making: apply fresh cell suspension and carry out steam droplets sheet, make karyomit(e) be dispersed, mark hybridization region;
(5) aging: in 50 ℃ of constant temperature roasters, 30min;
(6) sex change: be to mix at 1: 1 with No. 2 whole chromosome probes of people (Meta-systems company) ratio by the general centric fluorescent DNA probe of the direct mark of Fluorescein, at the probe mixed solution adding above the hybridization region having marked after two kinds of probes of 10 μ l mix, covered, by mounting rubber seal, close, be placed on roasting sheet machine 73 ℃ of sex change 3min;
(7) hybridization: slide is put into wet box, 37 ℃, spend the night;
(8) develop a film: at 72 ℃, use 0.4 * SSC 5min that develops a film; Then room temperature is used 2 * SSC (0.05%Tween-20), and 30s develops a film; DDW rinses, seasoning;
(9) redye: add 10 μ l DAPI, covered, lucifuge effect 10min;
(10) microscopy: observe karyomit(e) under fluorescent microscope and be blue, it is green that kinetochore is, and No. 2 whole chromosome takes on a red color, No. 2 karyomit(e)s of one bar long-armed with a long-armed generation transposition of No. 10 karyomit(e)s (referring to accompanying drawing 7).
Claims (7)
1. a detection method for radiation-induced rear cell dyeing volume morphing, its feature comprises the steps:
(1), prepare the general centric fluorescent DNA probe of the direct mark of fluorophor;
(2) the fluorescent DNA probe, step (1) being obtained and radiation-induced rear cell chromosome sample fluorescence in situ hybridization;
(3), under fluorescent microscope, detect kinetochore and chromosome morphology.
2. detection method according to claim 1, is characterized in that, the fluorophor described in step (1) is selected from any one in Cy3, Cy5 or Fluorescein.
3. detection method according to claim 1, is characterized in that, the DNA sequence dna of the fluorescent DNA probe that step (1) is described is from the α-satellite DNA in region, human chromosomal kinetochore.
4. detection method according to claim 3, is characterized in that, described step (1) comprises following two steps:
1) take Healthy People genomic dna as template, pcr amplification people α-satellite DNA fragment;
2) take step 1) α-satellite DNA fragment PCR products of obtaining is template, adds the dUTP with fluorophor in pcr amplification solution, obtains the general centric fluorescent DNA probe of the direct mark of fluorophor.
5. detection method according to claim 1, is characterized in that, described step (2) specifically comprises cell chromosome film-making, aging, sex change, hybridize, develop a film, redye and the step of microscopy.
6. detection method according to claim 5, is characterized in that, described in step (2), hybridizes and is specially: fluorescent DNA probe associating whole chromosome probe prepared by step (1) and radiation-induced rear cell chromosome sample hybridization.
7. detection method according to claim 6, is characterized in that, described whole chromosome probe is selected from any in people's whole chromosome probe.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105543342A (en) * | 2015-11-26 | 2016-05-04 | 集美大学 | Method for displaying centromeres and short arms of Larimichthys crocea |
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Patent Citations (3)
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| CN101886101A (en) * | 2009-05-12 | 2010-11-17 | 中山大学达安基因股份有限公司 | Method for preparing human chromosome 3 enumeration probe and application thereof |
| CN102517382A (en) * | 2009-05-12 | 2012-06-27 | 中山大学达安基因股份有限公司 | Human chromosome P16 gene detection kit and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105543342A (en) * | 2015-11-26 | 2016-05-04 | 集美大学 | Method for displaying centromeres and short arms of Larimichthys crocea |
| CN105543342B (en) * | 2015-11-26 | 2018-11-23 | 集美大学 | A method of display Larimichthys crocea centromere and galianconism |
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Application publication date: 20141029 |