CN106198358B - Chromosome sorting method based on " excitated type in quartz curette " flow cell sorter - Google Patents
Chromosome sorting method based on " excitated type in quartz curette " flow cell sorter Download PDFInfo
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- ZYVSOIYQKUDENJ-WKSBCEQHSA-N chromomycin A3 Chemical compound O([C@@H]1C[C@@H](O[C@H](C)[C@@H]1OC(C)=O)OC=1C=C2C=C3C[C@H]([C@@H](C(=O)C3=C(O)C2=C(O)C=1C)O[C@@H]1O[C@H](C)[C@@H](O)[C@H](O[C@@H]2O[C@H](C)[C@@H](O)[C@H](O[C@@H]3O[C@@H](C)[C@H](OC(C)=O)[C@@](C)(O)C3)C2)C1)[C@H](OC)C(=O)[C@@H](O)[C@@H](C)O)[C@@H]1C[C@@H](O)[C@@H](OC)[C@@H](C)O1 ZYVSOIYQKUDENJ-WKSBCEQHSA-N 0.000 claims abstract description 22
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1434—Optical arrangements
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N2015/1006—Investigating individual particles for cytology
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N2015/1028—Sorting particles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1434—Optical arrangements
- G01N2015/1438—Using two lasers in succession
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- Chemical & Material Sciences (AREA)
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- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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- Investigating Or Analysing Biological Materials (AREA)
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Abstract
The present invention provides a kind of chromosome sorting methods for being based on " excitated type in quartz curette " flow cell sorter.Further, the present invention provides the preparation methods of the chromosome sample for sorting.The method includes sorting the chromosome suspension after the Hoechst 33258 of preparation and chromomycin A3 dyeing on being based on " excitated type in quartz curette " flow cell sorter, UV and 444nm laser power used in it is respectively 50mW and 20mW, high-resolution two variables streaming caryogram is obtained, and sorts and has obtained the chromosome of high-purity.
Description
Technical field
The present invention relates to the technical fields of chromosome sorting.In particular it relates to be based on " excitated type in quartz curette "
The chromosome sorting method of flow cell sorter, and the method for the chromosome sample preparation for chromosome sorting.
Background technique
The chromosome of high-purity has been used for preparing chromosome painting probe [1,2], generate special DNA library [3,
4], microarray [5,6] etc..Streaming caryogram is the basis of DNA content and base composition difference based on different chromosomes
Upper and generation [7].33258 dyestuff of Hoechst is preferentially bound to the sequence of AT- enrichment, and chromomycin A3 (Chromomycin
A3) it is preferentially bound to the sequence [7] of GC- enrichment.The characteristics of both dyestuffs, can differentiate most human chromosomes, in addition to
Chromosome 9,10,11 and 12 clusters, because they have similar DNA content and base composition [7].
Chromosome sorting technology is exactly by chromosome Hoechst 33258 (abbreviation HO) and chromomycin A3 (abbreviation CA3)
Double fluorescein labels are carried out, the technology of specific chromosome is separated, purified, is enriched with by flow cell sorter.The present invention
In the chromosome sorting technology that is previously mentioned refer to the sorting technology of HO and CA3 double fluorescence labeling sample, referred to as " chromosome sorting ".
Current chromosome sorting " is excited in air by using MoFlo, Vantage SE, BD Influx etc.
Principle type " flow cell sorter is achieved.The discoveries such as Bee L.Ng are female thin with MoFlo selected by flow cytometry apoptosis human lymphocytes
The chromosome of born of the same parents system, the argon ion laser of two water coolings is UV laser (330-360nm) and blue-violet laser respectively
When (wavelength 458nm) is all set as 300mW, the two-dimentional streaming caryogram of preferable resolution ratio can be just obtained, to realize preferable sorting
Effect [8].The shortcoming of this " excitation principle type in air " equipment is that fluorescein is excited out in air, generation
Fluorescence signal energy loss is larger.It is glimmering in order to be detected in fluidic cell separation system since chromosome size is smaller
Optical signal needs that the marked fluorescein molecule in base is made to be excited out with highest energy, while avoiding fluorescence as far as possible
The loss of signal.Current chromosome sorting equipment is provided with high power laser, expensive.
At present using " excitation principle type in air " flow cell sorters such as MoFlo, Vantage SE, BD Influx
Chromosome sorting have that technical difficulty is big, not belongs to the irregular defect of routine techniques, detection level.Thus, in China
The country not yet really has developed so far.Chinese Academy of Sciences's Kunming animal institute, which is understood, according to us once utilized " excitation principle type in air "
Flow cell sorter (BD Vantage SE) is making an attempt at doing chromosome sorting technology, tentatively obtains human chromosome at that time
Streaming caryogram, but purpose chromosome is not sorted out always, the analytic process of chromosome is only stopped at, later never again
People carries out the further research of this technology.Therefore two-parameter chromosome sorting technology is not had developed really still at home,
So far it is classified as " blanking technique ".The country is more and more to the technical need, but is not being met always, therefore needs to develop at present
A kind of reliable, reproducible chromosome sorting method easy to operate, consistent.
For this purpose, we have done many-sided effort.Firstly, ordering particular arrangement in advance to prepare hardware condition
Type flow cell sorter.The core technology of BD Aria series of products is using " quartz curette flow cell " and " fully-reflected type signal
Detector ", fluorescent material are excited out in quartz curette, and loss of optical signal is small.Therefore it is presumed that if this Gao Ling
Sensitivity sorter may be more preferable applied to detection effect in chromosome sorting technology, and it is thin to be better than above-mentioned " excitated type in air "
Born of the same parents' sorter.Because the flow cell sorter of all commercialization BD Aria series does not have the configuration of chromosome sorting, so
We are special customized according to oneself application demand pre-designed optical path configuration, commission U.S. BD Bioscience company
A " excitated type in quartz curette " flow cell sorter-BD Aria SORP, belongs to the customized type in BD Aria series middle and high end
Product.The optical paths configuration such as the type, watt level, signal detector type of laser, optical filter combination is pressed in this equipment
According to our personalized thinking setting, it is different from the standard configurations type product such as Aria II, Aria III of commercialization.And " quartz curette flowing
Pond " and fully-reflected type signal detector principle are the patents of BD producer, have been widely used in all Aria series of products
In.
Second, We conducted the explorations of chromosome sorting sample preparation technology.The resolution ratio of streaming caryogram depends on
The detection sensitivity [8] of chromosome preparation and flow cytometer.Qualified sample is that chromosome sorting technology is successfully basic, but
Be chromosome sorted sample difficulty of preparation technology it is very big, it is domestic at present that qualified chromosome sorting sample can be provided almost without people
Product.Sample preparation procedure is more complex, prepares the step of largely free chromosome is most critical first, needs exist for groping thin
The a series of conditions such as born of the same parents' period " synchronization ", hypotonic, fluorescent marker, we establish by optimal conditions stablizes, efficiently contaminates
Colour solid preparation method.
Third, the exploration of chromosome sorting detection technique.The chromosome sample of preprepared qualification must be utilized,
It can start repeatedly to explore chromosome sorting operating technology, optical path, fluid path including complexity fine-tune process, final to obtain
Two variable streaming caryogram of chromosome, and be further purified, separate and be enriched with specifically according to the resolution ratio of every chromosome
Chromosome.We have also groped to most stable of instrument parameter to set, and have obtained in this equipment high-resolution
Two variable streaming caryograms, and realize the chromosome sorting technology of high-purity.
We utilize " excitated type in quartz curette " flow cell sorter to realize mouse and human cell's dye in the present invention
Colour solid sorting technology, the equipment that we use are different from " excitated type in air " equipment usual at present, for the first time with " in quartz curette
Excitated type " flow cell sorter (such as Aria SORP) realizes the sorting of chromosome.Our research is it has been proved that use
The chromosome detection sensitivity of Aria SORP is higher than " excitated type in air " equipment in the prior art, such as MoFlo or
Vantage SE, and our detection level has reached or even surmounts detection level in the prior art.Detection technique stabilization,
Reliably, reproducible.
In the present invention, we use low laser power, and wherein UV laser power is that 50mW, the 444nm of bluish violet are sharp
Light device power is 20mW, and only 1/6 and 1/15 laser power of " excitated type in air " equipment (300mW), avoids use
Expensive high power laser.And the chromosome resolution ratio detected has reached the peer-level with the prior art, even
It is higher, being successfully separated for No. 12 chromosomes of people is such as realized for the first time.
Summary of the invention
The present invention provides a kind of dyes for being based on " excitated type in quartz curette " flow cell sorter (such as Aria SORP)
Colour solid method for separating, wherein UV the and 444nm laser power difference that " excitated type in quartz curette " flow cell sorter uses
For 50mW and 20mW, and the two equal 300mW or more of laser power used in currently available technology；And in method of the invention
In, the excitation position of fluorescein is in " quartz curette ", and the fluorescein reported in the prior art excitation in " air ".The present invention
Confirm the chromosome sorting method high sensitivity of the invention based on " excitated type in quartz curette " flow cell sorter.
One kind be based on " excitated type in quartz curette " flow cell sorter chromosome sorting method, the method includes with
Lower step:
I) cell being harvested by centrifugation, suspension are incubated in hypotonic medium；
Ii) the suspension that centrifugation i) obtains, cell is resuspended in the polyamines separating liquid of frost；
Iii) acutely be vortexed concussion ii) obtain cell suspending liquid, centrifugation, filtering screening；
Iv) step iii) the chromosome suspension that obtains and Hoechst 33258 and chromomycin A3 be incubated overnight dyeing；
V) the chromosome suspension after dyeing is incubated on ice with sodium citrate and sodium sulfite；
Vi) the chromosome suspension after the dyeing for obtaining step v) is sorted based on " excitated type in quartz curette " fluidic cell
It is sorted on instrument, wherein UV the and 444nm laser power that " excitated type in quartz curette " flow cell sorter uses is respectively
50mW and 20mW.
Method according to the present invention, wherein the hypotonic medium contains 75mM KCl, 0.2mM spermine, the sub- essence of 0.5mM
Amine and 10mM MgSO47H2O, pH 8.0.
Method according to the present invention, wherein the polyamines separating liquid contains 15mM Tris, 2 mM EDTA, 0.5mM
EGTA, 80mM KCl, 3mM dithiothreitol (DTT), 0.25% Triton X-100,0.2mM spermine and 0.5mM spermidine, pH
7.5。
Method according to the present invention, wherein the Hoechst 33258 is 5 μ g/ml and chromomycin A3 is 50 μ g/m.
Method according to the present invention, wherein based on the UV laser in " excitated type in quartz curette " flow cell sorter
Detector selects 390nm optical filter, 444nm laser to select LP 475nm optical filter.
Method according to the present invention, wherein being set based on " excitated type in quartz curette " flow cell sorter design parameter
It sets as follows:
Nozzle: 70 μm of selection；
Laser power: UV laser power selects 50mW, 444nm laser power to select 20 mW；
Special filter plate: UV laser detector selects 390nm optical filter, 444nm laser to select LP 475nm optical filter；
Laser delay time and area factor: prolonging for UV laser and 444nm laser is precisely adjusted with rainbow microballoon
Slow time and area factor, so that the signal of respective channel reaches maximum；
The coefficient of variation (CV): making the CV value < 1.0 of UV and 444nm laser by hardware adjustments such as laser faculas, smaller
Better；
It obtains setting, " gating " of template: drawing following figure, FSC vs SSC scatter plot respectively, and draw P1 standards
The standby list for drawing a circle to approve chromosome monosomy, 33258 scatter plot of chromomycin A3 vs Hoechst, chromomycin A3 and Hoechst 33258
Parameter histogram；
The selection of threshold value: Hoechst 33258 and chromomycin A3 is selected to do double fluorescence thresholds respectively；
The gain of parameter and amplification mode select: FSC and SSC two parameter voltage selects logarithmic amplifier mode, chromomycin
A3 and 33258 two parameter voltage of Hoechst select linear amplification modes；
The adjusting of each parameter voltages；The voltage for adjusting FSC and SSC, is transferred to always FSC vs SSC scatter plot centre bit
It sets and most of chromosome particles occurs, big nucleus or adhesion body occurs in general upper right side, and small chromosome occurs in lower left
And fragment, and P1 sizes are adjusted, until drawing a circle to approve most of chromosome；
Drop delay: with Accudrop microballoon adjust delay time value, until sorting microballoon number reach 99% with
On；
The control of sample flow rate: chromosome sample loading rate adaptation to 300~1000/s；
Sorting mode: unicellular sorting mode is selected；
It sorts the preparation of receiver and sorts the collection of drop: preparing the dedicated load glass of pipe, FISH that inner wall prevents adhesion in advance
Piece or microwell plate receive the chromosome sorted out；
Separating purity verifying: fluorescence in situ hybridization is done with chromosome-specific probe, it is high-purity to verify the chromosome sorted
Degree and signal specificity.
On the other hand, the present invention provides a kind of chromosome sample preparation method for chromosome sorting, the methods
The following steps are included:
I) cell being harvested by centrifugation, suspension are incubated in hypotonic medium；
Ii) the suspension that centrifugation i) obtains, cell is resuspended in the polyamines separating liquid of frost；
Iii) acutely be vortexed concussion ii) obtain cell suspending liquid, centrifugation, filtering screening；
Iv) step iii) the chromosome suspension that obtains and Hoechst 33258 and chromomycin A3 be incubated overnight dyeing；
V) the chromosome suspension after dyeing is incubated on ice with sodium citrate and sodium sulfite.
The preparation method of chromosome sample according to the present invention, wherein the hypotonic medium contains 75mM KCl,
0.2mM spermine, 0.5mM spermidine and 10mM MgSO47H2O, pH 8.0.
The preparation method of chromosome sample according to the present invention, wherein the polyamines separating liquid contains 15mM Tris,
2mM EDTA, 0.5mM EGTA, 80mM KCl, 3mM dithiothreitol (DTT), 0.25%Triton X-100,0.2mM spermine and
0.5mM spermidine, pH 7.5.
The preparation method of chromosome sample according to the present invention, wherein the Hoechst 33258 be 5 μ g/ml and
Chromomycin A3 is 50 μ g/m.
Detailed description of the invention
Fig. 1: BD AriaTMThe configuration of SORP (6 laser, 18 color) detector.Wherein configure A (6b-3r-7v-2uv-0yg-
0bv), B (3b-3r-4v-2uv-3yg-3bv) is configured.
Fig. 2A is the two variable streaming caryogram of mouse chromosome based on " excitation principle in quartz curette " type Aria SORP；
Fig. 2 B is the two variable streaming caryogram [9] of mouse chromosome based on " excitation principle in air " type MoFlo.
Fig. 3 A-1 is the two variable streaming caryogram of human chromosome based on Aria SORP；
Fig. 3 A-2 is the amplified two variables streaming caryogram in human chromosome 9-12 cluster region in Fig. 3 A-1；
Fig. 3 B is the two variable streaming caryogram [10] of human chromosome based on Vantage SE.
Fig. 4 A is that the painting of the chromosome preparation in Fig. 3 A-2 after P1 groups of sortings contaminates probe in human peripheral blood mononuclear cell
(hPBMCs) the FISH result in；
Fig. 4 B is FISH knot of the painting dye probe of the chromosome preparation in Fig. 3 A-2 after P2 groups of sortings in hPBMCs
Fruit；
Fig. 4 C-4E respectively indicates the painting dye probe of the chromosome preparation in Fig. 3 A-2 after P1 groups of sortings in No. 12 chromosomes
Human embryonic stem cell B2-C7, B2-B8 of three-body and the FISH result for compareing hPBMCs.
Specific embodiment
1, the preparation for the chromosome sample of sorting
1) mouse boosting cell culture:
(1) a new 100mm Tissue Culture Dish is taken, 5ml lymphocyte separation medium Histopaque-1083 is added
(Sigma, St.Louis, MO), nylon membrane is placed on ware, is infiltrated film once with liquid the following.
(2) mouse spleen is transferred on film, is milled after being cut into several sections with scissors with 10ml syringe piston.
(3) after having milled, the lymphocyte separation medium with cell is transferred to 15ml centrifuge tube immediately.Carefully upper
Face adds 500 μ l RPMI, 1640 culture medium, and 800g is centrifuged 30min.
(4) intermediate buffy coat is sucked out to 50ml centrifuge tube with suction pipe, is added 1640 culture medium of RPMI, 300g from
Heart 10min.
(5) by isolated lymphocyte with 1x106Cells/ml is seeded in 1640 complete medium of RPMI, includes 10%
Fetal calf serum (FBS, Invitrogen, Carlsbad, CA), dual anti-(Penicillin and Streptomycin, Sigma,
St.Louis, MO), lipopolysaccharides (LPS, 50 μ g/ml, Sigma, St.Louis, MO) cultivates 48h.The autumn is added before harvesting cell
Water amide (Sigma, St.Louis, MO) extremely final concentration of 0.1 μ g/ml handles 3.5-6h.
2) human peripheral blood mononuclear cell (hPBMCs) cultivates:
(1) first by 5ml fresh human peripheric venous blood and 1640 culture medium 1:1 mixed diluting of 5ml RPMI.
(2) 5ml lymphocyte separation medium is added in 15ml centrifuge tube, then the human peripheric venous blood after 10ml is diluted
(10ml) is added slowly to lymphocyte separation medium HIstopaue 1077 (Sigma, St.Louis, MO) along tube wall in 45 degree of angles
In, 15-20min is centrifuged with 800g.
(3) after being centrifuged, the lymphocyte of blood and lymphocyte separation medium interface is sucked out, RPMI 1640 is added and cultivates
Base is centrifuged 10min with 400g.
(4) by isolated lymphocyte with 1x106Cells/ml is seeded in 1640 complete medium of RPMI, includes 10%
Fetal calf serum (FBS, Invitrogen, Carlsbad, CA), dual anti-(Penicillin and Streptomycin, Sigma,
St.Louis, MO), phytohemagglutin phytolectin (PHA, 5 μ g/ml, Sigma, St.Louis, MO) cultivates 72h.Add before harvesting cell
Enter amylose amide (Sigma, St.Louis, MO) to final concentration of 0.1 μ g/ml processing 3.5-6h.
3) prepared by chromosome
(1) 289g is centrifuged 5min and harvests cell.Cell precipitation be gently resuspended in 5ml hypotonic medium (75mM KCl,
0.2mM spermine, 0.5mM spermidine, 10mM MgSO47H2O.pH 8.0).
(2) at room temperature after hypotonic 15 minutes, the cell 289g of swelling is centrifuged 5min.Cell precipitation is gently resuspended in
In the polyamines separating liquid of 3ml frost (15mM Tris, 2mM EDTA, 0.5mM EGTA, 80mM KCl, 3mM dithiothreitol (DTT),
0.25%Triton X-100,0.2mM spermine, 0.5mM spermidine .pH 7.5), it is incubated for 10min on ice.
(3) be acutely vortexed cell suspending liquid concussion 20-40s.Chromosome suspension 201g is centrifuged 2min.By supernatant with 20
The filtering of μm granular membrane (Celltrics, Partec, M ü nster, Germany).
(4) chromosome suspension final concentration of 5 μ g/ml Hoechst 33258 (Sigma, St. Louis, MO), 50 μ g/
Ml chromomycin A3 (Sigma, St.Louis, MO), and 10mM MgSO47H2O (Sigma, St.Louis, MO), 4 DEG C of dyeing
Overnight.
(5) dye after chromosome suspension in streaming machine sorting before be added 10mM sodium citrate (Sigma,
St.Louis, MO) and 25mM sodium sulfite (Sigma, St.Louis, MO) be incubated at least 1h on ice.
2, chromosome sorting
Aria SORP flow cell sorter: being equipped with altogether six lasers, wherein being used for the laser of chromosome sorting
Device is three, is 488nm laser, maximum power 100mW respectively；UV laser 355nm, maximum power 100mW；444nm laser
Maximum power 40mW.In addition three lasers (405nm, 639nm, 561nm) are not explained in detail because unrelated with chromosome sorting.
All optical signals are received with optical fiber；It is " total reflection octagonal " that 488nm laser, which has been respectively adopted, in signal detector, UV laser,
The corresponding signal detector of 444nm laser is using " total reflection triangle "；Each photomultiplier tube (PMT) selects most sensitive-type
(the most sensitive-type selected from multiple PMT when equipment is installed by repetition test)；Spectroscope on every kind of signal detector and
The detector configuration that optical filter combination is detailed in Fig. 1.Crucial optical filter for chromosome sorting technology is respectively: the inspection of UV laser
Survey device is LP 390nm optical filter, and 444nm laser detector is LP 475nm optical filter.
Chromosome sorting is carried out with Aria SORP flow cell sorter: relating generally to what optical path adjusting and fluid path were adjusted
Process, design parameter setting and adjustment process are as follows:
(1) nozzle (noozle): 70 μm of selection；(2) laser power: UV laser power selects 50mW, 444nm laser power
Select 20mW；(3) special filter plate: UV laser detector selects 390nm optical filter, the selection LP 475nm filter of 444nm laser
Mating plate；(4) UV laser and 444nm precisely laser delay time (Time delay) and area factor: are adjusted with rainbow microballoon
The Time delay and area factor of laser, so that the signal of respective channel reaches maximum；(5) coefficient of variation (CV): pass through
The hardware adjustments such as laser facula make the CV value < of UV and 444nm laser less than 1.0, the smaller the better；(6) setting for template is obtained
It sets, " gating ": drawing following figure, FSC vs SSC scatter plot respectively, and it is mould to draw P1 preparation delineation chromosome monosomies, colors
The one-parameter of plain 33258 scatter plot of A3 vs Hoechst (P1 endoparticles of analysis), chromomycin A3 and Hoechst 33258 is straight
Fang Tu；(7) selection of threshold value: Hoechst 33258 and chromomycin A3 is selected to do double fluorescence thresholds respectively；(8) gain of parameter
It is selected with amplification mode；FSC and SSC two parameter voltage selects logarithm (log) amplification mode, chromomycin A3 and Hoechst
33258 two parameter voltages select linear (Lin) amplification mode；(9) adjusting of each parameter voltages；Adjust the electricity of FSC and SSC
Pressure is transferred to always the SSC scatter plot center FSC vs and most of chromosome particles occurs, and greatly thin occurs in general upper right side
There is small chromosome and fragment in karyon or adhesion body, lower left, and adjust P1 sizes, until drawing a circle to approve most of chromosome；
(10) drop delay (Drop delay): adjusting Dorp delay value with Accudrop microballoon, until sorting microballoon number
Reach 99% or more；(11) control of sample flow rate: chromosome sample loading rate adaptation to 300~1000/s；(12)
Sorting mode: unicellular sorting mode (single) is selected；(13) it sorts the preparation of receiver and sorts the collection of drop: is pre-
First prepare eppendorf (EP) pipe, FISH specialized glass slide or microwell plate that inner wall prevents adhesion, receives the dyeing sorted out
Body；(14) separating purity is verified: being FISH (fluorescence in situ hybridization) of chromosome-specific probe, is verified the chromosome sorted
High-purity and signal specificity.
3, result:
1) mouse chromosome of high-purity is obtained.
Fig. 2A and 2B [9] is respectively based on " excitation principle in quartz curette " type Aria SORP and " based on excimer in air
The two variable streaming caryogram of mouse chromosome of reason " type MoFlo.Two figures are respectively compared 13,15,16, No. 18 dyes in box
In colour solid and ellipse 8,9,10, the clarity of 12-14 chromosome because these chromosomes have similar DNA content
And base composition, so not being easily distinguishable most in mouse streaming caryogram, it can be seen that clarity is substantially better than in Fig. 2A
Fig. 2 B [9] illustrates that the sensitivity of the chromosome sorting based on " excitation principle in quartz curette " type is better than based on " excimer in air
Reason " type, the purity of higher the sorted out purpose chromosome of sensitivity are higher.
2) human chromosome of high-purity is obtained.
(partial enlargement i.e. centered on Fig. 3 A-1 box, 9-12 chromosome cluster are divided into P1 and P2 two by Fig. 3 A-1,3A-2
Group) and Fig. 3 B [10] respectively two variable streaming caryogram of human chromosome based on Aria SORP and Vantage SE.Because
Human chromosome 9-12 cluster has similar DNA content and base composition, so being difficult to differentiate between in streaming caryogram, such as Fig. 3 B
[10] shown in.But human chromosome 9-12 cluster can at least be divided by means of the present invention will left and right two groups (P1 and
P2), see Fig. 3 A-1,3A-2.It is a kind of dye that the FISH of the chromosome painting probe prepared by P1 and P2 groups, which has prompted P1 groups, left side,
Colour solid (Fig. 4 A), the right side P2 groups of mixtures (Fig. 4 B) for 3 kinds of chromosome.By special for human chromosome 9,10,11,12
P1 groups, the PCR prompt left side of primer is No. 12 chromosomes.Further dyed with the chromosome painting probe of P1 groups of preparations at No. 12
The human embryonic stem cell B2-C7 (Fig. 4 C) and B2-B8 (Fig. 4 B) of body three-body and the FISH for compareing hPBMCs (4C) are demonstrated
Left side P1 groups for No. 12 chromosomes, these results show the sensitivity of Aria SORP to be apparently higher than MoFlo or Vantage
SE。
4, conclusion
The present invention realizes chromosome sorting technology for the first time with " excitated type in quartz curette " flow cytometer.The present invention uses
" excitated type in quartz curette " type flow cytometer achieve the effect that " inexpensive object is more beautiful " compared with " excitated type in air ", even if
Laser power is lower, only with 1/6 to 1/15 exciting power of " excitated type in air " (300mW) equipment, can also reach
Ideal detection effect greatly reduces the investment of equipment, will save to researchers or testing service platform a large number of
Input.Chromosome Samples Preparation Technique established by the present invention has stable, reliable, repeatability with technology is operated the computer
The features such as good, easy to operate.The details for successfully preparing sorting chromosome sample is not only provided to researcher, can be obtained in a short time
Qualified chromosome sample is obtained, the time is saved, improves efficiency.In addition, that is established operates the computer method, guidance instrument operator
Member finds instrument regulation main points quickly, finds feeling, ensures the accuracy of detection, save the time.
In short, chromosome sorting technical thought novelty, technology that we establish have novelty, strong operability, instrument valence
It is honest and clean, detection sensitivity is high, it will bring good social benefit, have a extensive future in clinical and biomedical research application aspect.
Bibliography:
1.Rabbitts P,Impey H,Heppell-Parton A,Langford C,Tease C,et al.
(1995)Chromosome specific paints from a high resolution flow karyotype of the
mouse.Nat Genet 9:369-375.
2.Ng BL,Carter NP(2006)Factors affecting flow karyotype
resolution.Cytometry A 69:1028-1036.
3.Van Dilla MA,Deaven LL(1990)Construction of gene libraries for each
human chromosome.Cytometry 11:208-218.
4.Fantes JA,Green DK,Sharkey A(1994)Chromosome sorting by flow
cytometry.Production of DNA libraries and gene mapping.Methods Mol Biol 29:
205-219.
5.Fiegler H,Gribble SM,Burford DC,Carr P,Prigmore E,et al. (2003)
Array painting:a method for the rapid analysis of aberrant chromosomes using
DNA microarrays.J Med Genet 40:664-670.
6.Gribble SM,Fiegler H,Burford DC,Prigmore E,Yang F,et al. (2004)
Applications of combined DNA microarray and chromosome sorting
technologies.Chromosome Res 12:35-43.
7.Gribble SM,Ng BL,Prigmore E,Fitzgerald T,Carter NP(2009) Array
painting:a protocol for the rapid analysis of aberrant chromosomes using DNA
microarrays.Nat Protoc 4:1722-1736.
8.Ng BL,Carter NP(2010)Laser excitation power and the flow cytometric
resolution of complex karyotypes.Cytometry A 77:585-588.
9.Fengtang Yang VT,Bee Ling Ng,Nadezda Kosyakova,,Carter aNP(2009)
Generation of Paint Probes by Flow-Sorted and Microdissected
Chromosomes.Fluorescence In Situ Hybridization(FISH)–Application Guide:35-51.
10.Frey T,Houck DW,Shenker BJ,Hoffman RA(1994)Bivariate flow
karyotyping with air-cooled lasers.Cytometry 16:169-174.
Claims (6)
1. one kind is based on the chromosome sorting method of " excitated type in quartz curette " flow cell sorter, the method includes following
Step:
I) cell being harvested by centrifugation, suspension are incubated in hypotonic medium；
Ii) the suspension that centrifugation i) obtains, cell is resuspended in the polyamines separating liquid of frost；
Iii) acutely be vortexed concussion ii) obtain cell suspending liquid, centrifugation, filtering screening；
Iv) step iii) the chromosome suspension that obtains and Hoechst 33258 and chromomycin A3 be incubated overnight dyeing；
V) the chromosome suspension after dyeing is incubated on ice with sodium citrate and sodium sulfite；
Vi) the chromosome suspension after the dyeing for obtaining step v) is based on " excitated type in quartz curette " flow cell sorter
Sorting, wherein UV the and 444nm laser power that " excitated type in quartz curette " flow cell sorter uses be respectively 50mW and
20mW。
2. according to the method described in claim 1, wherein the hypotonic medium contains 75mM KCl, 0.2mM spermine, the sub- essence of 0.5mM
Amine and 10mM MgSO47H2O, pH 8.0.
3. according to the method described in claim 1, wherein the polyamines separating liquid contains 15mM Tris, 2mM EDTA, 0.5mM
EGTA, 80mM KCl, 3mM dithiothreitol (DTT), 0.25%Triton X-100,0.2mM spermine and 0.5mM spermidine, pH 7.5.
4. according to the method described in claim 1, wherein the Hoechst 33258 is 5 μ g/ml and chromomycin A3 is 50 μ g/
m。
5. method according to claim 1-4, wherein being based on " excitated type in quartz curette " flow cell sorter
In UV laser detector selection 390nm optical filter, 444nm laser select LP 475nm optical filter.
6. method according to claim 1-4, wherein being based on " excitated type in quartz curette " flow cell sorter
Design parameter is provided that
Nozzle: 70 μm of selection；
Laser power: UV laser power selects 50mW, 444nm laser power to select 20mW；
Special filter plate: UV laser detector selects 390nm optical filter, 444nm laser to select LP 475nm optical filter；
Laser delay time and area factor: the delay time of UV laser and 444nm laser is precisely adjusted with rainbow microballoon
And area factor, so that the signal of respective channel reaches maximum；
Coefficient of variation CV: making the CV value < 1.0 of UV and 444nm laser by the hardware adjustments of laser facula, the smaller the better；
It obtains setting, " gating " of template: drawing following figure, FSC vs SSC scatter plot respectively, and draw P1 preparation circles
The one-parameter for determining chromosome monosomy, 33258 scatter plot of chromomycin A3 vs Hoechst, chromomycin A3 and Hoechst 33258 is straight
Fang Tu；
The selection of threshold value: Hoechst 33258 and chromomycin A3 is selected to do double fluorescence thresholds respectively；
The gain of parameter and amplification mode select: FSC and SSC two parameter voltage selects logarithmic amplifier mode, chromomycin A3 with
33258 two parameter voltage of Hoechst selects linear amplification modes；
The adjusting of each parameter voltages: the voltage of FSC and SSC is adjusted, FSC vs SSC scatter plot center is transferred to always and goes out
Existing most of chromosome particles, and P1 sizes are adjusted, until drawing a circle to approve most of chromosome；
Drop delay: adjusting delay time value with accudorp microballoon, until sorting microballoon number reaches 99% or more；
The control of sample flow rate: chromosome sample loading rate adaptation to 300~1000/s；
Sorting mode: unicellular sorting mode is selected；
Sort the preparation of receiver and sort the collection of drop: prepare in advance the pipe that inner wall prevents adhesion, FISH specialized glass slide or
Microwell plate receives the chromosome sorted out；
Separating purity verifying: doing fluorescence in situ hybridization with chromosome-specific probe, verify the chromosome high-purity sorted and
Signal specificity.
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