CN105483255A - TOP2A gene detection probe, preparation method thereof and reagent kit - Google Patents
TOP2A gene detection probe, preparation method thereof and reagent kit Download PDFInfo
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Abstract
The invention relates to a TOP2A gene detection probe and a preparation method thereof. The method includes the following steps that a selected BAC clone is at least one of RP11-1152A10 and CTD-3217C5, or a selected BAC clone is at least one of RP11-737D6, CTD-3087O22, RP11-48O10 and CTD-2134K5, a plasmid is extracted from the clone to obtain DNA of the plasmid, and quantification is conducted; marking is carried out with fluorescein. The invention further discloses a reagent kit provided with the TOP2A gene detection probe. By obtaining the optimal TOP2A detection probe through screening, signal line counting is accurate and quick, and result repeatability is good. Defects of TOP2A mutation detection in clinic are made up for, more patients benefited from targeted drug can be screened more easily, the survival rate of the patients is increased, and overall life time is prolonged.
Description
Technical field
The invention belongs to biotechnology, particularly relate to TOP2A gene test probe and preparation method thereof and test kit.
Background technology
TOP2A gene is DNA topoisomerase II α (TopoisomeraseIIalpha, TOPII α) encoding gene, the biological action of DNA topoisomerase, one is the superhelix state of regulable control DNA and the catenane state of knotting or the DNA that unhitches, thus affects indirectly intracellular nucleic acid metabolic process; Two is participate in those directly need interrupt and the cell processes reconnecting DNA molecular chain, the restructuring of DNA, repairs, transcribes and reproduction process.There is patient's poor prognosis of TOP2A gene unconventionality, without recurrence short survival, especially patient's prognosis of TOP2A genetically deficient is poorer.Gene amplification prompting tumour has the possibility of recurrence, or the curative effect in long term declines.
Anthracene nucleus medicament is the foundation stone of Postoperative Adjuvant Chemotherapy in Breast Cancer, research confirms TOP2A gene [Brase relevant to anthracycline curative effect, JC, Schmidt, M, Fischbach, T, Sultmann, H, Bojar, H, Koelbl, H (2010) ERBB2andTOP2Ainbreastcancer:acomprehensiveanalysisofgene amplification, RNAlevels, andproteinexpressionandtheirinfluenceonprognosisandpredi ction.ClinCancerRes16:pp.2391-2401, Fountzilas, G, Christodoulou, C, Bobos, M, Kotoula, V, Eleftheraki, AG, Xanthakis, I (2012) TopoisomeraseIIalphageneamplificationisafavorableprognos ticfactorinpatientswithHER2-positivemetastaticbreastcanc ertreatedwithtrastuzumab.JTranslMed10:pp.212, BartlettJMS, McConkeyCC, MunroAF, etal.PredictingAnthracyclineBenefit:TOP2AandCEP17-NotOnlybutAlso [J] .JournalofClinicalOncology, 2015:JCO.2013.54.7869.], TOP2A gene appearance changes and tumor recurrence risk, survival of patients is correlated with.
Therefore the TOP2A state of correct detection and evaluation mammary cancer is most important.The TOP2A assignment of genes gene mapping is in No. 17 karyomit(e) 17q21 regions, and the abnormal conditions of TOP2A are divided into amplification and disappearance two kinds of situations, and FISH detects to make for both of these case and clearly judges.
Fluorescence in situ hybridization (FluorescenceinsituhybridizationFISH) is a kind of nonradioactivein situhybridization technology grown up on the basis of original radioactive in situ hybridization technology phase late 1980s.This technology current has been widely used in the structural research of animal-plant gene group, the analysis of variance of karyomit(e) fine structure, viral infection assays, mankind's antenatal diagnosis, cancer genetics and genome evolution research and has treated many fields.The ultimate principle of FISH is probe with known labeling nucleic acid, according to the principle of base complementrity, carries out anisogamy with single-chain nucleic acid unknown in material to be checked, form the heteroduplex nucleic acid that can be detected.Because DNA molecular is linearly arrange along the karyomit(e) longitudinal axis on chromosome, thus can probe directly and karyomit(e) carry out hybridizing thus specific gene located on chromosome.Compared with traditional radio-labeling in situ hybridization, fluorescence in situ hybridization has fast, detection signal is strong, hybrid specificities is high and can the feature such as multiple staining, is therefore subject to common concern in molecular cytogenetics field.
Hybridize probe used roughly can to classify three classes: 1) the special repetitive probe of karyomit(e), the such as probe of α satellite, satellite III class, its hybridization target position is often greater than 1Mb, not containing interspersed repeat sequence, be combined closely with target position, hybridization signal is strong, is easy to detect; 2) whole chromosome or chromosomal region specific probe, it is made up of extremely different nucleotide fragments on section a certain on item chromosome or karyomit(e), special large fragment can be obtained by the karyomit(e) be cloned in phage and plasmid; 3) specificity position probe, is made up of one or several cloned sequence.
The fluorescein-labelled of probe can adopt directly and the method for indirect labelling.Indirect labelling adopts biotin-labelled DNA probe, fluorescein avidin is associated with lotus root or Streptavidin detects after hybridization, Avidin-Biotin-luciferin complexes can also be utilized simultaneously, fluorescent signal be amplified, thus the fragment of about 500bp can be detected.And direct labelling method is by fluorescein directly and probe nucleotide or phosphopentose skeleton covalent attachment, or when nick-translation label probe, fluorescein ribonucleoside triphosphote is mixed.Direct labelling method step when detecting is simple, and Clinical practice is convenient.
And at present TOP2A gene FISH method is detected, also lack the detection kit that specificity is high.
Summary of the invention
An object of the present invention is to provide a kind of TOP2A gene test probe and preparation method thereof, and prepared probe can be used for detecting TOP2A gene appearance, namely detects the finger copy number change of TOP2A gene, has good specificity.
The technical scheme realizing above-mentioned purpose is as follows.
A preparation method for TOP2A gene test probe, comprises the following steps:
(1) choosing BAC clone is at least one in RP11-1152A10, CTD-3217C5, or chooses BAC clone at least one in RP11-737D6, CTD-3087O22, RP11-48O10, CTD-2134K5;
(2) respectively plasmid is extracted to clone, obtain plasmid DNA, quantitatively;
(3) use fluorescein-labelled plasmid DNA, the fluorescein that the plasmid DNA of different sources marks is identical, to obtain final product.
Wherein in an embodiment, described BAC clone is RP11-1152A10 and CTD-3217C5.
Wherein in an embodiment, described BAC clone is RP11-737D6, CTD-3087O22, RP11-48O10 and CTD-2134K5.
Wherein in an embodiment, mark fluorescent element selects fluorescence dye known in the art, and preferably, fluorescein is
fITC,
rhodamine, TexasRed,
dEAC.
Wherein in an embodiment, the mark of gene probe can adopt method of the prior art by corresponding fluorescein-labelled on double-strandednucleic acid, described method includes but not limited to: random priming, nick translation etc., mark gene probe can use commercially available nick translation labelling kit and/or random primer labelling kit, the NickTranslationKit of preferred abbott and/or Roche company.Step of the present invention (3) preferably adopts random priming, nick-translation method to carry out fluorescein-labelled to plasmid DNA.
Wherein in an embodiment, the temperature of described mark is 24 DEG C-26 DEG C, and the time of mark is 4-6 hour.
Another object of the present invention is to provide a kind of TOP2A gene detecting kit.
Realize this object technical scheme as follows.
A kind of TOP2A gene detecting kit, includes above-mentioned TOP2A gene test probe.
Wherein in an embodiment, No. 17 karyomit(e)s included for internal control differentiate probe (CSP17) probe, and this discriminating probe is not identical with the color of the fluorescein of TOP2A gene test probe mark.
Wherein in an embodiment, also include the COTHumanDNA for closed tumor-necrosis factor glycoproteins, and DAPI counterstain.
The present invention has following beneficial effect:
1. the present invention is by screening optimum TOP2A gene test probe and combination thereof, adopts FISH (FluorescenceIn-SituHybridization) method to detect TOP2A gene copy number,
2. preferably to clone detection specificity good, highly sensitive in the present invention.And by adjustment mark temperature and time, limited length probe is about 500bp, improves hybridization efficiency and reduces hybrid context.
3. signal-count row accurately, fast, and result is reproducible; Supplement the deficiency of clinical middle TOP2A abrupt climatic change, be conducive to screening the patient benefiting from targeted drug more, improve patient with breast cancer's survival rate and Overall survival.
4. by TOP2A test kit of the present invention, understand TOP2A state from gene level to change, multi-signal type list reveals the tumour cell genetic diversity of solid tissue, can be implemented in the application in the field such as oncobiology, cytogenetics, help each molecular marker of comprehensive evaluation, the auxiliary clinical targeted therapy medication of mammary cancer and treatment plan are selected.
Accompanying drawing explanation
Fig. 1 is be the schematic diagram of detection probes sequence in embodiment 1.
Fig. 2 is human peripheral culturing cell sheet FISH detected result figure in embodiment 1.
Fig. 3 is breast cancer tissue's sample FISH detected result figure in embodiment 4, and wherein, detection signal type is that 2R2G, TOP2A gene increases.
Fig. 4 is breast cancer tissue's sample FISH detected result figure in embodiment 4, and wherein, detection signal type is 6-12R2G, TOP2A gene amplification.
Embodiment
For the ease of understanding the present invention, will be described more fully the present invention below.The present invention can realize in many different forms, is not limited to embodiment described herein.On the contrary, provide the object of these embodiments be make the understanding of disclosure of the present invention more comprehensively thorough.
The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to the condition that manufacturer advises.Various conventional chemical reagent used in embodiment, is commercially available prod.
Unless otherwise defined, all technology used in the present invention and scientific terminology are identical with belonging to the implication that those skilled in the art of the present invention understand usually.The object of the term used in specification sheets of the present invention just in order to describe specific embodiment, is not used in restriction the present invention.Term "and/or" used in the present invention comprises arbitrary and all combinations of one or more relevant Listed Items.
The preparation of embodiment 1TOP2A gene test probe
This implements the preparation method of described TOP2A detection probes, comprises the following steps.
Select the clone comprising goal gene TOP2A and two terminal sequences, as shown in Figure 1.
GSPTOP2A comprises two groups, comprises the first probe, the second probe, the 3rd probe and four point probe respectively. and table specific as follows, it is bought in InvitrogenRP11BAC and CTDBAC clone bank.Two groups of detection probes are below divided to prepare respectively.
TOP2Achr17:38,544,773-38,574,202,29,430bp
| Probe groups 1 | BAC | Insert Fragment start-stop position |
| First probe | RP11-1152A10 | chr17：38376940…38546337(169Kb) |
| Second probe | CTD-3217C5 | chr17：38521566…38730400(209Kb) |
| Probe groups 2 | BAC | Insert Fragment start-stop position |
| 3rd probe | RP11-737D6 | chr17：38257742…38440899(183Kb) |
| Four point probe | CTD-3087O22 | chr17：38430841…38564776(134Kb) |
| 5th probe | RP11-48O10 | chr17：38564771…38724970(160Kb) |
| 6th probe | CTD-2134K5 | chr17：38716958…38834072(117Kb) |
(2) GSPTOP2A gene test probe preparation: the PlasmidMaxiKit using Qiagen company, the working method required to specifications carries out ultralow copy extraction of plasmid DNA respectively to different B AC clone, quantitative to plasmid DNA by the absorbancy measuring 260nm and 280nm place; Adopt autoclaved ultrapure water to dilute for 200ng/ul, adopt the centrifuge tube packing of 1.5ml, 2 kinds or 4 kinds of plasmid DNA mixing finally will obtaining ,-20 DEG C of sealings are preserved.
(3) carry out fluorescent mark by nick translation method to plasmid DNA cocktail, the fluorescein of often kind of probe mark is Spectrum-Orange.Adopt the NickTranslationKit of abbott, by following scheme, under strict lucifuge condition, prepare PCR reaction system on ice.
10×NTbuffer5ul
dTTP(1mM)0.4ul
d3TPs(1mM)1ul
Spectrum-OrangedUTP0.6ul
NT enzyme 10ul
Plasmid DNA (100ng/ul) each 2.5ul
Add H
2o is 50ul to cumulative volume.。
Join rear concussion mixing, mark 5 hours at 25 DEG C, then 80 DEG C hatch 10 minutes inactivators.Getting 5ul uses 2% sepharose to do electrophoresis, there is the band of disperse at about 500bp.
Alcohol settling carried out to marked product and concentrate, in 1.5ml centrifuge tube, adding sodium-acetate and dehydrated alcohol by following scheme successively, lucifuge, on ice preparation:
Marked product 45ul
Sodium-acetate (3mol/L) 5ul
Dehydrated alcohol 125ul
Mixing to be placed in-70 DEG C of refrigerators at least 2 hours, and centrifugal 30 minutes of 4 DEG C of 13000rpm, carefully remove supernatant, do not stir precipitation, add 70% ethanol of 1ml, 4 DEG C 13000 revs/min centrifugal 15 minutes, carefully removes supernatant, do not stir precipitation, and lucifuge is dry.Use 1ul purified water dissolution precipitation, obtain GSPTOP2A gene probe, lucifuge ,-20 DEG C of storages.
(4) GSPTOP2A gene probe checking: use two groups of probes respectively, drips sheet for sample to be tested with mankind's proper splitting lymphocyte in mid-term and carries out the checking (detection method with reference to prior art and embodiment 3) of two groups of probes.Comprise mid-term or interphase chromosome DNA in culturing cell, during fluorescence in situ hybridization, chromosomal DNA shows as discernible karyomit(e) or nucleus in form.Coming to the same thing of two groups of probes, as shown in Figure 2: the FISH results of hybridization figure of Metaphase Chromosome.Karyomit(e) 17q21 position display red fluorescent can be seen in figure, (No. 17 karyomit(e)s differentiate probe to internal control probe CSP17, can mark No. 17 karyomit(e)s, purchased from SE17, D17Z1 (KBI-20017, KREATECH) shows green fluorescence.In figure, visible TOP2A gene probe signal becomes clear, and can be observed sensitivity, specificity 100% in human peripheral culturing cell sheet on Metaphase Chromosome; Use paraffin sample chips to carry out hybridization check, can clearly record TOP2A gene copy number.
Embodiment 2:TOP2A gene detecting kit preparation method
TOP2A gene detecting kit includes TOP2A hybridization solution and DAPI counterstain two components, wherein TOP2A hybridization solution comprise GSPTOP2A gene probe described in embodiment 1 (being respectively two groups of detection probes, corresponding two kinds of test kits), CSP17 probe (No. 17 Chromosome Identification probes), for hybridizing the buffer composition of environment (promoting hybridization), closing the COTHumanDNA etc. of tumor-necrosis factor glycoproteins.DAPI counterstain is mainly used in the cell after hybridizing and redyes, and DAPI wherein can be combined with DNA, makes nucleus demonstrate blue-fluorescence, and the counterstain containing Ursol D can keep the stable of fluorescence.
Concrete formula is as follows:
(1) hybridization solution preparation
(2) DAPI counterstain preparation
The Ursol D of 10mg is dissolved in the PBS of 1ml, regulates pH to be 9.0, adds 9ml glycerine, repeatedly shake mixing ,-20 DEG C of storages.The DAPI solution (0.1mg/ml) getting 2.5 μ l is dissolved in that 1ml is anti-to fade in liquid, repeatedly shakes mixing ,-20 DEG C of airtight preservations of lucifuge under lucifuge condition.
(3) finished product assembling
| Ingredient names | Specification/10test | Quantity |
| Hybridization solution | 100 μ l/ manage | 1 pipe |
| DAPI counterstain | 100 μ l/ manage | 1 pipe |
| Specification sheets | 1 part |
The detection method of embodiment 3:TOP2A gene detecting kit
1, slide pre-treatment
1.1 slides are put into the roasting sheet of 65 ± 5 DEG C of thermostat containers and are spent the night;
1.2 take out slides, to put it in dimethylbenzene room temperature dewaxing 15 minutes;
1.3 take out slides, then to put it in another cylinder dimethylbenzene room temperature and continue dewaxing 15 minutes;
1.4 take out slides, then to put it in dehydrated alcohol room temperature 10 minutes, remove residual dimethylbenzene;
1.5 taking-up slide, then put it into each 3 minutes of 100%, 90%, 70% graded ethanol room temperature rehydration;
1.6 taking-up slide, then to put it in purified water room temperature washing 3 minutes, draw excessive moisture with lint-free paper handkerchief;
1.7 take out slides, then to put it in purified water 100 ± 5 DEG C and boil sheet 25 minutes (section is placed horizontally in container, and sample faces up);
1.8 take out slide, and room temperature is dried;
1.9 being faced up by slide puts on the top of the shelf, drip appropriate stomach en-reaction solution in sample areas, digest 5 ~ 15 minutes;
Surplus liquid gets rid of by 1.10, to put it in room temperature 2 × SSC 5 minutes;
1.11 take out slides, then to put it in another cylinder room temperature 2 × SSC 5 minutes;
1.12 taking-up slide, then it is put into room temperature 70% successively, 90%, 100% each 3 minutes of graded ethanol dehydration;
1.13 take out slide, and room temperature is dried.
The pepsic reaction times needs to be determined by trial test.Can use with batch preparation sample chips carry out trial test by described method, usually with 5 minutes for interval time.Such as, testing digestion time is respectively 5 minutes, 10 minutes and 15 minutes, after completing " slide pre-treatment ", under light field, can use 10 × or 20 × object lens tissues observed Digestive States; Or directly carry out DAPI to redye, carry out Digestive States judgement.
2, sample and the same time variation of probe (lucifuge operation)
2.12.1 from-20 ± 5 DEG C of refrigerators, the hybridization solution in detection kit described in embodiment 2 is taken out, concussion mixing, brief centrifugation;
2.22.2 the hybridization solution adding 10 μ l, to hybridising region, covers rapidly 18 × 18mm cover glass, and light pressure makes hybridization solution be uniformly distributed, and avoids producing bubble;
2.32.3 with rubber glue along cover glass edge mounting, cover the position that cover glass contacts with slide glass completely;
2.42.4 slide is put into hybridization instrument, moistening in situ hybridization instrument humidity bar, inserts wet bar, cover hybridization instrument upper cover, " Denat & Hyb " program is set, sex change 85 DEG C 5 minutes, hybridize 37 DEG C 10 ~ 18 hours.(if amixia instrument, can use alternative instrument, and as Thermostatic platform carries out sex change, Electric heat oven/or water-bath are hybridized, and should be noted that temperature is accurate and keeps hybridization humidity).
3, post-hybridization washing and redying (lucifuge operation)
3.1 first 30 minutes of washings, by the washing lotion I prepared, washing lotion II, put into the water-bath of 37 ± 1 DEG C, measure to guarantee that temperature is suitable;
3.2 close hybridization instrument power supply, are taken out by slide, tear rubber glue gently off, remove cover glass and (if cover glass is difficult to remove, can puts it in washing lotion I and slightly rock, be beneficial to it and come off;
3.3 slide puts into 37 ± 1 DEG C of washing lotion I (2 × SSC) 10 minutes;
3.4 taking-up slide, then to put it in 37 ± 1 DEG C of washing lotion II (0.1%NP-40/2 × SSC) 5 minutes;
3.5 take out slides, in room temperature 70% ethanol 3 minutes;
3.6 take out slide, dark place seasoning slide;
3.7 room temperatures, drip the cover glass of 10 μ lDAPI counterstains to 22 × 22mm, slide glass target area down, is put down gently on cover glass, is gently pressed, and avoids producing bubble, in the dark deposits, to be seen.
Above-mentioned cited reagent all prepares (often kind of reagent volume is 40ml) in circular staining jar, and each staining jar can put at most 5 sections.Non-solution at room temperature, needs to shift to an earlier date preheating reaction reagent to assigned temperature before operation starts.In washing process, within 2 ~ 3 minutes, staining jar can be rocked gently in interval, improve washing effect.
4, interpretation of result
Fluorescence associated and DAPI need observe with suitable filter block.Wherein, CSP17 probe display green; GSPTOP2A probe is danger signal.
4.1 use suitable filter, find under 40 × object lens, count under 100 × object lens;
The focal length that 4.2 adjustment are suitable, has clear and definite concept to signal and background; Signaling point is because being positioned at cell; When extracellular exists fluorescent signal point, note distinguishing with Intracellular signals point, preferably can avoid this region and count;
The 4.3 several tumour cell regions of pan, select at least 4 regions having fine core to demarcate, require that nuclear boundary is complete, DAPI even dyeing, core zero lap, CSP17 probe (green point) signal is clear;
4.4 analyze from the upper left corner of selected zone, from left to right sweep, observe multiple visual field;
The requirement of 4.5 tissue counts:
A. tumor tissues (before FISH detects, using HE staining section to carry out controlled observation) is only counted
B. avoid at necrotic zone and the unclear area count of nuclear boundary
C. the core of subjective discrimination is needed not count
D. bypass signal is weak and do not have the nuclear counting of signal specific or high background;
4.6 go to 100 × object lens, adjusting focal length, find all signaling points in the different levels of core;
4.7 at each core inside counting signaling point; All signaling points in each core are found in focusing, counts two kinds of signals in a region, and only counting often kind of color has 1 or more FISH signal, does not have signal or only has a kind of core of color signal not count; Record the total cellular score (signal is normal and abnormal) observed;
4.8 method of counting
At 5 tumor regions clearly, amount to GSPTOP2A (redness) and CSP17 (green) signal in several 40 ~ 100 neoplastic cell nucleis, count the number of signals of GSPTOP2A and CSP17 in single nucleus respectively.
Embodiment 4:TOP2A gene detecting kit Clinical practice is evaluated
Use two groups of detection probes described in embodiment 1, detection kit described in embodiment 2, to 20 parts of clinical samples (it is made a definite diagnosis through pathology detection, specifically sees the following form), detects.According to the detection method duplicate detection 3 times of embodiment 3, result conforms to, detected result reproducible; The detection consistence of two kinds of probe combinations is good.With commercial goods reagents ratio comparatively, detected result is completely the same, the specificity of reagent and highly sensitive.Fig. 3 and Fig. 4 is the detected result figure of probe groups 1.The result of probe groups 2 and coming to the same thing of probe groups 1, figure omits.Fig. 3 is negative sample detected result, and type signal type is 2R2G, and result is judged as that TOP2A gene increases; Fig. 4 is positive sample detected result, and signal type is 2 ~ 6R/2G, and result is judged as TOP2A gene amplification.Danger signal in figure shows GSPTOP2A, and green shows CSP17 (for locating No. 17 chromosome centromere probes).
In the present invention, a kind of probe respectively in each use TOP2A gene also can realize corresponding detection, and concrete outcome omits, but probe combinations uses relatively, the use of combination probe, and detection signal can be better.Probe length is longer in theory, and the fluorescent signal brightness obtained during actual detection is brighter, but because may relate to more polygene sequence, the signal complicacy possibility obtained increases, and also strengthens detecting the difficulty realized.BAC clone total length for the group 1 of TOP2A gene and the detection probes of group 2 of the present invention is respectively: 353Kb and 576Kb, is the nucleic acid mixture comprising TOP2A gene and two terminal sequences thereof.
Contriver finds in probe checking of the present invention, and longer detection probes obtains stronger fluorescent signal really, and also obtain identical result in the detection validation of clinical sample.Therefore, in the design of fluorescent probe, luminance signals can be increased by proper extension fluorescent probe length, but specifically how to combinationally use, the certain technical difficulty existed, realize good detected result, except the experience in design, also need by clinical sample checking assessing signal type difference.
Each technical characteristic of described embodiment can combine arbitrarily, for making description succinct, all possible combination of each technical characteristic in above-described embodiment is not all described, but, as long as the combination of these technical characteristics does not exist contradiction, be all considered to be the scope that this specification sheets is recorded.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore be construed as limiting the scope of the patent.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (9)
1. a preparation method for TOP2A gene test probe, is characterized in that, comprises the following steps:
(1) choosing BAC clone is at least one in RP11-1152A10, CTD-3217C5, or chooses BAC clone at least one in RP11-737D6, CTD-3087O22, RP11-48O10, CTD-2134K5;
(2) respectively plasmid is extracted to BAC clone, obtain plasmid DNA, quantitatively;
(3) use fluorescein-labelled plasmid DNA, the fluorescein that the plasmid DNA of different sources marks is identical, to obtain final product.
2. the preparation method of TOP2A gene test probe according to claim 1, is characterized in that, described BAC clone is RP11-1152A10 and CTD-3217C5; Or described BAC clone is RP11-737D6, CTD-3087O22, RP11-48O10 and CTD-2134K5.
3. the preparation method of TOP2A gene test probe according to claim 1, it is characterized in that, described fluorescein is Alexa
fITC, Alexa
rhodamine, TexasRed, pacific
or DEAC.
4. the preparation method of TOP2A gene test probe according to any one of claim 1-3, is characterized in that, step (3) adopts random priming or nick-translation method to carry out fluorescein-labelled to plasmid DNA.
5. the preparation method of TOP2A gene test probe according to claim 4, it is characterized in that, the temperature of described mark is 24 DEG C-26 DEG C, and the time is 4-6 hour.
6. the TOP2A gene test probe that the preparation method according to any one of claim 1-5 obtains.
7. a TOP2A gene detecting kit, is characterized in that, includes TOP2A gene test probe according to claim 6.
8. TOP2A gene detecting kit according to claim 7, is characterized in that, No. 17 karyomit(e)s also included for internal control differentiate probes, and this discriminating probe is not identical with the color of the fluorescein of TOP2A gene test probe mark.
9. TOP2A gene detecting kit according to claim 7 or 8, is characterized in that, also includes the COTHumanDNA for closed tumor-necrosis factor glycoproteins, and DAPI counterstain.
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| CN201511030984.9A CN105483255A (en) | 2015-12-30 | 2015-12-30 | TOP2A gene detection probe, preparation method thereof and reagent kit |
| PCT/CN2016/105717 WO2017114010A1 (en) | 2015-12-30 | 2016-11-14 | Top2a gene detection probe, preparation method therefor, and test kit |
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| CN201511030984.9A CN105483255A (en) | 2015-12-30 | 2015-12-30 | TOP2A gene detection probe, preparation method thereof and reagent kit |
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| WO2017114010A1 (en) * | 2015-12-30 | 2017-07-06 | 广州安必平医药科技股份有限公司 | Top2a gene detection probe, preparation method therefor, and test kit |
| WO2017114011A1 (en) * | 2015-12-30 | 2017-07-06 | 广州安必平医药科技股份有限公司 | Her-2 gene and/or top2a gene detection probe, preparation method therefor, and test kit |
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Cited By (2)
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| WO2017114010A1 (en) * | 2015-12-30 | 2017-07-06 | 广州安必平医药科技股份有限公司 | Top2a gene detection probe, preparation method therefor, and test kit |
| WO2017114011A1 (en) * | 2015-12-30 | 2017-07-06 | 广州安必平医药科技股份有限公司 | Her-2 gene and/or top2a gene detection probe, preparation method therefor, and test kit |
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