CN105524990A - HER-2 gene and / or TOP2A gene detection probe and preparation method and kit - Google Patents
HER-2 gene and / or TOP2A gene detection probe and preparation method and kit Download PDFInfo
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Abstract
The present invention relates to a HER-2 gene and / or TOP2A gene detection probe and a preparation method thereof, and the method comprises the following steps: selecting BAC aiming at HER-2 gene to clone into RP11-1021P11, RP11-62N23 and RP11-1044P23, or selecting BAC to clone into RP11-98J2, RP11-1065L22 and CTD-2327I15; and / or selecting BAC aiming at TOP2A gene to clone into RP11-1152A10 and CTD-3217C5, or selecting BAC to clone into RP11-737D6, CTD-3087O22, RP11-48O10 and CTD-2134K5; obtaining plasmid DNA; and marking. The present invention also discloses a kit which includes the HER-2 gene and / or TOP2A gene detection probe. The best HER-2 gene and TOP2A gene detection probe is screened, so that a signal counting line is accurate and rapid, and results are well reproducible.
Description
Technical field
The invention belongs to biotechnology, particularly relate to HER-2 (ERBB2) gene and/or TOP2A gene test probe and preparation method thereof and test kit.
Background technology
HER-2 (ERBB2) gene has the amplification of gene and the overexpression of albumen in the patient with breast cancer of 20%-30%.HER-2 is the important prognosis/predictor of the mammary cancer of generally acknowledging at present, and in multivariate analysis result, HER-2 is the independent prognostic factor of tumor recurrence and Overall survival length.The Breast Cancer Infiltration of the HER-2 positive is strong, and the disease free survival phase is short, poor prognosis.Herceptin (Trastuzumab) be applicable to HER-2 overexpression and gene amplification mammary cancer.The superhelix state of TOP2A generegulation control DNA, participates in cell processes directly, and is the target enzyme of anthracycline antibiotics, there is patient's poor prognosis of TOP2A gene unconventionality, and 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.TOP2A gene appearance all has definite meaning in the response situation and medication effect of indication chemotherapy of tumors.
Therefore correct detect and evaluation mammary cancer HER-2 gene and TOP2A gene appearance most important.
The HER-2 assignment of genes gene mapping is in No. 17 karyomit(e) 17q12 regions, and the exception of HER-2 is amplification; 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 HER-2 gene and/or TOP2A gene FISH are 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 HER-2 gene and/or TOP2A gene test probe and preparation method thereof, prepared probe can be used for detecting HER-2 gene and TOP2A gene appearance, namely detect the finger copy number change of HER-2 gene and TOP2A gene, there is good specificity.
The technical scheme realizing above-mentioned purpose is as follows.
A preparation method for HER-2 gene and/or TOP2A gene test probe, comprises the following steps:
(1) the BAC clone chosen for HER-2 gene is at least one in RP11-1021P11, RP11-62N23, RP11-1044P23, or chooses BAC clone at least one in RP11-98J2, RP11-1065L22, CTD-2327I15; And/or the BAC clone chosen for TOP2A gene is at least one in RP11-1152A10, CTD-3217C5, or choose 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 for same gene marks is identical, and the color of the fluorescein marked with the detection probes for TOP2A gene for HER-2 gene is not identical, to obtain final product.
Wherein in an embodiment, the described BAC clone for TOP2A gene is RP11-1152A10 and CTD-3217C5.
Wherein in an embodiment, the described BAC clone for TOP2A gene is RP11-737D6, CTD-3087O22, RP11-48O10 and CTD-2134K5.
Wherein in an embodiment, the BAC clone for HER-2 gene is RP11-1021P11, RP11-62N23 and RP11-1044P23.
Wherein in an embodiment, the described BAC clone for TOP2A gene is RP11-98J2, RP11-1065L22 and CTD-2327I15.
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 HER-2 gene and TOP2A gene detecting kit.
Realize this object technical scheme as follows.
A kind of HER-2 gene and/or TOP2A gene detecting kit, include above-mentioned HER-2 gene and/or 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 with HER-2 gene.
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 HER-2 gene and/or TOP2A gene test probe and combination thereof, adopts FISH (FluorescenceIn-SituHybridization) method to detect HER-2 gene and TOP2A gene copy number,
2. preferably to clone detection specificity good, highly sensitive in the present invention.And by adjustment mark time and temperature, limited length probe is about 500bp, improves hybridization efficiency and reduce hybrid context.
3. signal-count row accurately, fast, and result is reproducible; By detecting HER-2, TOP2A copy number and No. 17 karyomit(e)s and assess simultaneously, be conducive to determining treatment plan, screen the patient with breast cancer benefiting from targeted drug more, improve survival and Overall survival.
4. by HER-2 gene of the present invention and TOP2A test kit, HER-2 gene and the change of TOP2A state is understood from gene level, 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, assist Prognosis in Breast Cancer judgement and clinical targeted therapy medication and treatment plan to select.
Accompanying drawing explanation
Figure 1A is be the schematic diagram of HER-2 gene test probe sequence in embodiment 1.
Figure 1B is be the schematic diagram of TOP2A gene test probe sequence in embodiment 1.
Fig. 2 is human peripheral culturing cell sheet HER-2 gene and TOP2A gene test probe 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 2R2G2A, HER-2 gene increases, and TOP2A gene increases.
Fig. 4 is breast cancer tissue's sample FISH detected result figure in embodiment 4, and wherein, detection signal type is cluster R cluster G2A, HER-2 gene and TOP2A gene generation coamplification.
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 1HER-2 gene and TOP2A gene test probe
A kind of preparation method of HER-2 detection probes (GSPHER-2), comprises the following steps:
Colony screening: select the clone comprising goal gene HER-2 and two terminal sequences, as shown in Figure 1A.
GSPHER-2 comprises two groups, below divides two groups of detection probes to prepare respectively.
HER-2chr17:37,856,254-37,884,915,28,662bp
The preparation method of described TOP2A detection probes (GSPTOP2A), comprises the following steps:
Colony screening: select the clone comprising goal gene TOP2A and two terminal sequences, as shown in Figure 1B.
GSPTOP2A comprises two groups, table specific as follows, and 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
(2) GSPHER-2 and 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, finally by the plasmid DNA mixing of 2 kinds or 3 kinds or 4 kinds respective combination of the corresponding HER-2 gene that obtains or TOP2A gene ,-20 DEG C of sealings are preserved.
(3) by nick translation method, fluorescent mark is carried out to plasmid DNA cocktail, fluorescein for often kind of probe mark of GSPHER-2 gene is Spectrum-Orange, and the fluorescein of often planting probe mark for GSPTOP2A gene is Spectrum-Green.Adopt the NickTranslationKit of abbott, by following scheme, under strict lucifuge condition, prepare PCR reaction system on ice.
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 respectively 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.GSPHER-2 and GSPTOP2A gene probe is verified: use hybridization solution prepared by HER-2 probe groups 1+TOP2A probe groups 1 respectively, the hybridization solution of HER-2 probe groups 2+TOP2A probe groups 2 preparation is reagent to be detected, uses mankind's proper splitting lymphocyte in mid-term to drip sheet and carries out probe checking (detection method reference example 3).Culturing cell comprises mid-term or interphase chromosome DNA, and during fluorescence in situ hybridization, chromosomal DNA shows as discernible karyomit(e) or nucleus in form.As shown in Fig. 2 (being applied to the experimental result of HER-2 probe groups 1+TOP2A probe groups 1): the FISH results of hybridization figure of Metaphase Chromosome.In figure, red color visible signal shows GSPHER-2, and green shows GSPTOP2A, and cyan signal shows CSP17 (for locating No. 17 chromosome centromere probes, being purchased from D17Z1SpectrumAquaProbe, article No.: 06J38-017).In figure, visible HER-2/TOP2A/17 chromosome 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, clearly can record three target copy numbers.Other corresponding probe confirmatory experiment result is identical with the experimental result of HER-2 probe groups 1+TOP2A probe groups 2, and figure omits.
Embodiment 2:HER-2 gene and TOP2A gene detecting kit preparation method
HER-2 gene and TOP2A gene detecting kit include HER-2/TOP2A hybridization solution and DAPI counterstain two components, and wherein HER-2 and TOP2A hybridization solution comprises the combination of one group of GSPTOP2A gene probe described in embodiment 1 and one group of GSPHER-2 gene probe.HER-2 and TOP2A gene has two groups of detection probes respectively, two groups of two kinds of genes can arbitrarily be combined, in the present embodiment, described HER-2 gene and TOP2A gene detecting kit are four kinds, be respectively: the combination of HER-2 (group 1)+TOP2A (group 1), the combination of HER-2 (group 2)+TOP2A (group 2), the combination of HER-2 (group 1)+TOP2A (group 2), the combination of HER-2 (group 2)+TOP2A (group 1), CSP17 probe (No. 17 Chromosome Identification probes, be purchased from D17Z1SpectrumAquaProbe, article No.: 06J38-017), for hybridizing the buffer composition of environment (promoting hybridization), close 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:HER-2 gene and 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 take out slide, then put it into each 3 minutes of 100%, 90%, 70% graded ethanol room temperature rehydration;
1.6 take out slides, 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;
Slide faces up by 1.9 to be put on the top of the shelf, drips appropriate stomach en-reaction solution, digest 5 ~ 15 minutes in sample areas;
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 take out slide, then it are 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 use 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 slides put into 37 ± 1 DEG C of washing lotion I (2 × SSC) 10 minutes;
3.4 take out slides, 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, danger signal shows GSPHER-2, and green shows GSPTOP2A, and cyan signal shows CSP17.
4.1 use suitable filter, find, count under 100 × object lens under 40 × 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 (cyan signaling 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:HER-2 gene and TOP2A gene detecting kit Clinical practice are evaluated
Use HER-2 gene and TOP2A gene test probe combinations described in embodiment 1, to 20 parts of clinical samples, (it makes a definite diagnosis through pathology detection 4 kinds of detection kit (combination of HER-2 (probe groups 1)+TOP2A (probe groups 1), the combination of HER-2 (probe groups 2)+TOP2A (probe groups 2), the combination of HER-2 (probe groups 1)+TOP2A (probe groups 2), the combination of HER-2 (probe groups 2)+TOP2A (probe groups 1)) described in embodiment 2, specifically see the following form), detect respectively.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 composite reagent box detected result of HER-2 (probe groups 2)+TOP2A (probe groups 2), shown in Fig. 3, breast carcinoma paraffin wax embedded tissue samples FISH detected result, signal type shows as 2R2G2A, result is judged as HER-2/TOP2A gene no abnormality seen, and No. 17 karyomit(e)s are normal; In Fig. 4, breast cancer tissue sample FISH detected result, signal type shows as cluster R cluster G2 ~ 6A, and result is judged as HER-2/TOP2A gene coamplification, No. 17 many bodies of karyomit(e).The detected result of the combination of other different two kinds of gene probes is identical, and figure omits.
The detected result of the combination of HER-2 (probe groups 1)+TOP2A (probe groups 2), the combination of HER-2 (probe groups 2)+TOP2A (probe groups 1), come to the same thing with shown in upper table, because of the reason of length, concrete outcome omits.
In the present invention, for HER-2 gene and TOP2A gene, a kind of probe of each use also can realize corresponding detection respectively, 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 for the probe groups 1 of HER-2 gene and the detection probes of probe groups 2 of the present invention clones its length and is respectively: 510Kb and 569Kb, is the nucleic acid mixture comprising HER-2 gene and two terminal sequences thereof.BAC clone total length for the probe groups 1 of TOP2A gene and the detection probes of probe groups 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.
Know from above-mentioned laboratory test results, after molecular marker detection is carried out to these samples, molecule parting can be carried out according to detected result to sample, according to the meaning of Testing index, for clinical treatment formulation, medication selection and Outcome measure.
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 (10)
1. a preparation method for HER-2 gene and/or TOP2A gene test probe, is characterized in that, comprise the following steps:
(1) the BAC clone chosen for HER-2 gene is at least one in RP11-1021P11, RP11-62N23, RP11-1044P23, or chooses BAC clone at least one in RP11-98J2, RP11-1065L22, CTD-2327I15; And/or the BAC clone chosen for TOP2A gene is at least one in RP11-1152A10, CTD-3217C5, or choose 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 for same gene marks is identical, and for HER-2 gene and/or the color of fluorescein that marks for the detection probes of TOP2A gene not identical, to obtain final product.
2. the preparation method of HER-2 gene and/or TOP2A gene test probe according to claim 1, it is characterized in that, BAC clone for HER-2 gene is RP11-1021P11, RP11-62N23 and RP11-1044P23, or is RP11-98J2, RP11-1065L22 and CTD-2327I15 for BAC clones.
3. the preparation method of HER-2 gene and/or TOP2A gene test probe according to claim 1, it is characterized in that, described BAC clone for TOP2A gene is RP11-1152A10 and CTD-3217C5, or is RP11-737D6, CTD-3087O22, RP11-48O10 and CTD-2134K5 for the described BAC clone of TOP2A gene.
4. the preparation method of HER-2 gene and/or TOP2A gene test probe according to claim 1, it is characterized in that, BAC clone for HER-2 gene is RP11-1021P11, RP11-62N23 and RP11-1044P23, and is RP11-1152A10 and CTD-3217C5 for the described BAC clone of TOP2A gene.
5. the preparation method of HER-2 gene and/or TOP2A gene test probe according to claim 1, it is characterized in that, BAC for HER-2 gene clones as BAC clone is RP11-98J2, RP11-1065L22 and CTD-2327I15, and is RP11-737D6, CTD-3087O22, RP11-48O10 and CTD-2134K5 for the described BAC clone of TOP2A gene.
6. the preparation method of HER-2 gene and/or TOP2A gene test probe according to claim 1, it is characterized in that, described fluorescein is Alexa
fITC, Alexa
rhodamine, TexasRed,
dEAC.
7. the preparation method of HER-2 gene and/or TOP2A gene test probe according to any one of claim 1-6, it is characterized in that, step (3) adopts random priming or nick-translation method to carry out fluorescein-labelled to plasmid DNA, the temperature of described mark is 24 DEG C-26 DEG C, and the time is 4-6 hour.
8. the HER-2 gene that the preparation method according to any one of claim 1-7 obtains and/or TOP2A gene test probe.
9. HER-2 gene and/or a TOP2A gene detecting kit, is characterized in that, includes HER-2 gene according to claim 8 and/or TOP2A gene test probe.
10. HER-2 gene and/or TOP2A gene detecting kit according to claim 9, it is characterized in that, No. 17 karyomit(e)s also included for internal control differentiate probe, and this discriminating probe is not identical with the color of the fluorescein of TOP2A gene test probe mark with HER-2 gene; Also include the COTHumanDNA for closed tumor-necrosis factor glycoproteins, and DAPI counterstain.
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