CN103409504A - FISH (fluorescence in situ hybridization) probe, kit and detection method for detecting Her2 (human epidermal growth factor receptor 2) gene free from repetitive sequence - Google Patents
FISH (fluorescence in situ hybridization) probe, kit and detection method for detecting Her2 (human epidermal growth factor receptor 2) gene free from repetitive sequence Download PDFInfo
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Abstract
The invention belongs to the field of biotechnology, and discloses a FISH (fluorescence in situ hybridization) probe, a kit and a detection method for detecting Her2 (human epidermal growth factor receptor 2) gene free from repetitive sequence. The Her2 gene is used as a template to perform polymerase chain reaction on non-repetitive sequence in the Her2 gene, the amplification product is DNA (deoxyribonucleic acid) fragments with 350-800 different base pairs, the repetitive sequence in the Her2 gene is eliminated so as to form the product free from repetitive sequence; after the product is in fluorescence labeling, the Her2 gene free from repetitive FISH is obtained for the Her2 gene FISH detection. The Her2 gene FISH probe obtained by the invention can eliminate the repetitive sequence, the non-specific background signal of the Her2 gene FISH probe can be obviously reduced, and the specificity of the FISH probe is improved.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of FISH probe, test kit and detection method thereof that does not contain tumor-necrosis factor glycoproteins for detection of the Her2 gene.
Background technology
Mammary cancer is the common cancer that the women ranks the first.The recent statistics data presentation of U.S. CA:A Cancer Journal for Clinicians magazine announcement in 2011, the U.S. estimated to have 230480 routine women to suffer from mammary cancer in 2011, account for 30% of women's de novo malignancy, first of rank women's Cancer Mortality.Metropolitan statistics shows that mammary cancer is the modal malignant tumour of China women equally in China Beijing, Shanghai, Tianjin etc., and sickness rate is ascendant trend year by year.Breast cancer disease is because illustrating not yet fully, but many research datas show, removing outside native factor of mammary cancer, also with the long-time stimulus of endogenous or exogenous estrogen: playing an important role to breast cancer expressed in crossing of estrogenic activity and EGF-R ELISA (Her2).
Proto-oncogene ErbB-2 (human epidermal growth factor receptor-2, Her2) gene, it is the c-erbB-2 gene, be positioned on karyomit(e) 17q12-21.32, the coding relative molecular mass is 185000 transmembrane receptor sample albumen, has tyrosine kinase activity.HER2 is the important Prognosis in Breast Cancer judgement factor, and Her2 crosses the mammary cancer of expressing or increasing, and its clinical characters and biological behaviour have Special Manifestations, and the mammary cancer for the treatment of pattern, curative effect and prognosis and other types makes a big difference.Target therapeutic agent Trastuzumab for the Her2 Data mining is also expressed or amplification based on crossing of Her2 gene, and clinical use Trastuzumab medicine is effective to Her2 gene overexpression or amplification patient, invalid without Her2 gene overexpression person patient.
At present, for detection of the gold standard of the types such as gene rearrangement, fusion, amplification, be fluorescence in situ hybridization technique clinically.Fluorescence in situ hybridization (fluorescence in situ hybridization, FISH) is a kind of on-radiation molecular cytogenetics technology grown up on the basis of radioactive in situ hybridization technology in late 1980s.Utilize specific nucleic acid after fluorescein-labelled as probe, after with target DNA, hybridizing, by fluorescent microscope, directly take individual cells again and count as the object of observation, can in 36 hours, realize qualitative, quantitatively and positioning analysis to chromosome segment to be measured.Yet fluorescence in situ hybridization probe is mainly used the artificial chromosome preparation, these artificial chromosomes comprise yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) and phage artificial chromosome (PAC) etc., owing in artificial chromosome, there being a lot of tumor-necrosis factor glycoproteinss, these tumor-necrosis factor glycoproteinss constantly occur in whole genome, thereby cause the fluorescence in situ hybridization probe prepared to have a lot of tumor-necrosis factor glycoproteinss, these tumor-necrosis factor glycoproteinss also can produce fluorescence after probe and the hybridization of non-target DNA, thereby produce nonspecific fluorescent signal, greatly reduce specificity and sensitivity that FISH detects.Therefore, this area active demand exploitation Her2 gene do not contain tumor-necrosis factor glycoproteins FISH probe reagent box, be used to specificity and the sensitivity of the fluorescence in situ hybridization experiment that improves the Her2 gene.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of FISH probe, test kit and detection method thereof that does not contain tumor-necrosis factor glycoproteins for detection of the Her2 gene is provided.
A kind of FISH probe that does not contain tumor-necrosis factor glycoproteins for detection of the Her2 gene, described FISH DNA probe is to take Her2 gene and kinetochore (STR) sequence to be template, by 27 pairs of primer sets or the nucleotide sequence that has identity with primer sets, adopting the polymerase chain technology to carry out pcr amplification reaction prepares, in described 27 pairs of primer sets, the 1-26 group is Her2 gene primer sequence, the 27th pair of primer sets is kinetochore STR aligning primer, described primer sets is (wherein 1,2,3 ... 27 is the primer title, F is upstream primer, and R is downstream primer):
1 F：5' GGTTGGAATGAGTAAGAAGTAGC 3'
1 R：5' TAGGAATTTTCTAAAGGGGAGAC 3'
2 F：5' AGGTTAATGGTGGAAGTGGG 3'
2 R：5' ACACTCTACTATTAACATATGC 3'
3 F：5' TTTACTTTGGCTGCTGTTGC 3'
3 R：5' ATATTTTCGGATTTGGTATAGG 3'
4 F：5' TGGCTGTAGGTCAGAGTGGC 3'
4 R：5' AAGGATAAGGTGGAGTTTTGT 3'
5 F：5' TGGGTGATGGAATGATCTGG 3'
5 R：5' ACGAGTCTCCGAACAAAAGG 3'
6 F：5' AAAAGAAAACCAATGGGACAG 3'
6 R：5' CCCACGCCAACATAAATACAC 3'
7 F：5' TCCTCCCTCTTTGTCCTTATC 3'
7 R： 5' AAAGTGCCCAGTTGAACTTGTT 3'
8 F： 5' CTTTGTGGCAGCAGTTTCGT 3'
8 R： 5' AGAGCAGATGGGTCAGAGGAC 3'
9 F： 5' GTGGTGGGACTCAAAGACGG 3'
9 R： 5' AGGAAATGGGTGGTGTAGGC 3'
10F： 5' CCCTAGAAGGTGATGCTGATG 3'
10R： 5' ATATGCTCCCATTTACAGAAAC 3'
11F： 5' CCAGCCCACCCTGTCCTAT 3'
11R： 5' TGCCCACTCTTACCCATCAA 3'
12F： 5' CCAGCCCACCCTGTCCTA 3'
12R： 5' TTCTGTCTCCTGCCATCCC 3'
13F： 5' TGCCTGACCTCAGCGTCTT 3'
13R： 5' GGTGGGTGTTATGGTGGATG 3'
14F： 5' ACACCCACCTCTGCTTCGT 3'
14R： 5' TGCCTTCACTCAAACCCTTT 3'
15F： 5' TGGGTGAAGAGCAAGGGTG 3'
15R： 5' GAGTGGGTGCAGTTGATGG 3'
166F：5' CCAGGAGCATGGCGAAAA 3'
16R： 5' AAGAGCCCCAGAAAGACCC 3'
17F： 5' CTCACTTTCCAGAACCTT 3'
17R： 5' TTATTTGACTCGACCTAA 3'
18F： 5' CCTTCATTGCCCTTCACTC 3'
18R： 5' CCTAGCCATCTTTCCTTGC 3'
19F： 5' ATGTCTTCAAAGTTCTGGTG 3'
19R： 5' AGATAGTGCATCCTGGGA 3'
20F： 5' GGGTGGTGAAGGATGTTTG 3'
20R： 5' TAGTCTAGGTTTGCGGGAGT 3'
21F： 5' GCCCTATGGCTGCCTCTTA 3'
21R： 5' CTCTGCTCCTTGGTCCTTCAC 3'
22F：5' TATGCCCTATGGCTGCCTCT 3'
22R：5' TGGTCCTTCACCTAACCTTGC 3'
23F：5' GGTCTGTCTCCTGGCATCAC 3'
23R：5' ACATCCTGGTCCCCTTTCA 3'
24F：5' ACAGGCACCGCAGCTCAT 3'
24R：5' CGCCACTCTGGGGACAAG 3'
25F：5' TCTCCACTGGCACCCTCC 3'
25R：5' GACGACCCCATTCTTCCCT 3'
26F：5' CTTCAAAGGGACACCTACGG 3'
26R：5' CAAAGCCTGGATACTGACACC 3'
27F：5' GGCACGGTGGCTCACGCCTGTAAT 3'
27R：5' GGGAGGCTAAGTTCCTTGTTCG 3'。
In such scheme, hold and all insert the universal tag sequence in 5 ' of described 27 pairs of primer sets:
CGCTTTACTGACGCGTTGC, can carry out the secondary amplification of PCR product, thereby can amplify the PCR product for the universal tag sequence, reduce production costs, and improves the probe preparation efficiency.
A kind ofly comprise the above-mentioned test kit that does not contain the FISH probe of tumor-necrosis factor glycoproteins for detection of the Her2 gene.
In the mentioned reagent box, described FISH probe is the double-colored probe of fluorescence in situ hybridization, the double-colored probe of described FISH is pulvis, the double-colored probe of described FISH comprises Her2 red fluorescence probe and kinetochore green fluorescence probe, and the mass ratio of described Her2 red fluorescence probe and kinetochore green fluorescence probe is 1:1.
In the mentioned reagent box, described FISH probe is the double-colored probe of fluorescence in situ hybridization, the double-colored probe of described FISH is liquid reagent, the double-colored probe of described FISH is to obtain by Her2 red fluorescence probe and kinetochore green fluorescence probe are dissolved in to the 2x sodium citrate buffer solution, wherein the concentration of Her2 red fluorescence probe is 5ng/ul(w/v), the concentration of kinetochore green fluorescence probe is 5ng/ul(w/v), described 2x sodium citrate buffer solution is with sodium-chlor 8.8g, Trisodium Citrate 4.4g, deionized water 400ml configuration forms.
In the mentioned reagent box, also comprise 4', 6-diamidino-2-phenylindone (DAPI) is redyed liquid.
A kind of mentioned reagent box that utilizes, to the method that the Her2 gene detects, comprises the steps: that specifically (1) is fixed on tissue to be detected on slide, and the tissue to be measured on slide is carried out to pre-treatment; (2) tissue to be measured on probe and slide is carried out to the sex change hybridization of nucleotide sequence; (3) after hybridization, slide is washed; (4) use and to redye liquid the hybridization zone on slide is redyed; (5) use the hybridization zone of fluorescence microscope slide.
In such scheme, the pre-treatment of the described tissue to be detected of step (1) is: (1) is placed in paraffin organization section to be measured on fragmentation, slide is placed under 65 ℃ and toasted 12～16 hours again, then with dimethylbenzene, dewax, subsequently slide is placed in to 100%(vt% successively), 85%(vt%), 70%(vt%) the ethanol gradient solution each 2 minutes, immersed again in deionized water 3 minutes, and with thieving paper, drew unnecessary moisture after taking-up; (3) with acid S-WAT, process and organized slide 20～30 minutes under 50 ℃, under 90 ℃, use water treatment slide 30 minutes, use again the rinsing of 2 * sodium citrate buffer solution, then slide is immersed in Proteinase K solution to 10～20 minutes under 37 ℃, use subsequently the rinsing of 2 * sodium citrate buffer solution; (4) with the fixing slide of formaldehyde, then slide is placed in to the ethanol gradient solution dewaters, finally by naturally drying, and be heated to 56 ℃, standby.
In such scheme, the nucleotide sequence sex change hybridization of the described probe of step (2) and tissue to be measured is: (1) gets each 1ul of probe reagent, 2x sodium citrate buffer solution 7ul, deionized water 1ul, be mixed to get the probe mixed solution, sex change is 5 minutes under 78 ℃ of conditions, then is placed in rapidly under 45 ℃ of conditions, takes out before hybridization; (2) slide after pretreatment was immersed in 78 ℃ of methane amide/2 * sodium citrate buffer solutions to denaturing treatment 30 minutes, be placed in again the 70%(vt% of-20 ℃ of precoolings), 85%(vt%), 100%(vt%) the ethanol gradient solution carries out processed, then by after the slide seasoning, be placed in 45 ℃ of condition preheatings; (3) the probe mixing drop after above-mentioned sex change is regional in slide hybridization, be placed under 45 ℃ of conditions and hybridized 12～16 hours.
In such scheme, the described slide washing of step (3) is: remove cover glass, slide is placed in to 0.3%(wt%) Nonidet P40/0.4 * sodium citrate buffer solution, rinsing 2 minutes; Again fragmentation is placed in to 0.1%(wt%) Nonidet P40/2 * sodium citrate buffer solution, rinsing 30 seconds; Finally slide is placed in to 70%(vt%) ethanol, rinsing 3 minutes.
In prior art, because in Her2 gene FISH probe, containing a large amount of tumor-necrosis factor glycoproteinss, before use, at first need with Cot-1DNA reagent, the tumor-necrosis factor glycoproteins in sample DNA to be sealed, this enclosed experiment is not only consuming time, and reagent used is expensive; Her2 gene FISH probe prepared by the present invention does not contain tumor-necrosis factor glycoproteins, in use, without with Cot-1DNA reagent, carrying out enclosed experiment, easy to operate, and economical and efficient has shortened detection time of test kit greatly.
Beneficial effect of the present invention:
(1) preparation process of FISH probe of the present invention, combine nucleic acid hybridization technique, fluorescent mark technology, fluorescence microscopy imaging technique, the gold standard that the FISH probe detects as gene break, fusion, amplification etc., and detection sensitivity is high;
(2) prepared by the present invention does not contain the hybridization identification of tumor-necrosis factor glycoproteins FISH probe to the Her2 nucleic acid molecule, has accuracy high, and specificity is good, and the low advantage of false positive;
(3) production cost of middle probe of the present invention and test kit is low, is convenient to large-scale production;
(4) simple to operate, the safety of test kit of the present invention, reduced the use of the expensive reagent such as Cot-1 DNA reagent, the anti-DigiTAb of rhodamine, economical and efficient simultaneously;
(5) compared with prior art, the detection method of test kit of the present invention, without using Cot-1 DNA to seal the sample gene, easy to operate, detection speed is fast, can in the 12-24 lab scale, complete the hybridization check experiment.
The accompanying drawing explanation
Fig. 1 is that the Her2 gene is used repetition and non repetitive sequence schematic diagram, the wherein XXX drawn after Repeat Masking database analysis ... XXX represents tumor-necrosis factor glycoproteins.
Fig. 2 is 1-27 primer pair PCR product electrophorogram, wherein the template of 1-26 group is BAC clone's Her2 gene DNA, the template of the 27th group is kinetochore STR sequence plasmid, in figure, molecular weight marker thing (M) is DNA ladder (DNA ladder), 1,2,3 ... 27 products that increase for corresponding primer pair separately.
Fig. 3 does not contain tumor-necrosis factor glycoproteins Her2 fluorescence probe in situ hybridization figure, and wherein template is the mammary cancer sample.It in figure, is 1. kinetochore green fluorescence probe mark DNA fragmentation; 2. be Her2 red fluorescence labeled dna fragment; 3. be Her2 red fluorescence probe mark DNA fragmentation, show as the gene form that increases in a large number.In Fig. 3, A is that the Her2 gene is normal, and the quantity ratio of the DNA of the DNA of centromeric probe mark and Her2 probe mark is normal; B is the amplification of Her2 gene unconventionality, 1. with Her2 probe mark DNA fragmentation ratio 3., can find out that Her2 increases in a large number from green centromeric probe labeled dna fragment shown in white arrow figure.
Embodiment
In order to understand better the present invention, below in conjunction with accompanying drawing, embodiment, further illustrate content of the present invention, but content of the present invention not only is confined to following example.
Synthesizing of primer sets: by on-line analysis database Repeat Masking(
Http:// www.girinst.org/) the Her2 gene is analyzed, obtain repetition and the non repetitive sequence (as shown in Figure 1) of Her2 gene, non repetitive sequence is imported to primer-design software Primer5 and carries out design of primers, design primer extension product at 350～800bp, and synthesize following 26 pairs of primers by the method for synthetic:
1 F：5' GGTTGGAATGAGTAAGAAGTAGC 3'
1 R：5' TAGGAATTTTCTAAAGGGGAGAC 3'
2 F：5' AGGTTAATGGTGGAAGTGGG 3'
2 R： 5' ACACTCTACTATTAACATATGC 3'
3 F： 5' TTTACTTTGGCTGCTGTTGC 3'
3 R： 5' ATATTTTCGGATTTGGTATAGG 3'
4 F： 5' TGGCTGTAGGTCAGAGTGGC 3'
4 R： 5' AAGGATAAGGTGGAGTTTTGT 3'
5 F： 5' TGGGTGATGGAATGATCTGG 3'
5 R： 5' ACGAGTCTCCGAACAAAAGG 3'
6 F： 5' AAAAGAAAACCAATGGGACAG 3'
6 R： 5' CCCACGCCAACATAAATACAC 3'
7 F： 5' TCCTCCCTCTTTGTCCTTATC 3'
7 R： 5' AAAGTGCCCAGTTGAACTTGTT 3'
8 F： 5' CTTTGTGGCAGCAGTTTCGT 3'
8 R： 5' AGAGCAGATGGGTCAGAGGAC 3'
9 F： 5' GTGGTGGGACTCAAAGACGG 3'
9 R： 5' AGGAAATGGGTGGTGTAGGC 3'
10F： 5' CCCTAGAAGGTGATGCTGATG 3'
10R： 5' ATATGCTCCCATTTACAGAAAC 3'
11F： 5' CCAGCCCACCCTGTCCTAT 3'
11R： 5' TGCCCACTCTTACCCATCAA 3'
12F： 5' CCAGCCCACCCTGTCCTA 3'
12R： 5' TTCTGTCTCCTGCCATCCC 3'
13F： 5' TGCCTGACCTCAGCGTCTT 3'
13R： 5' GGTGGGTGTTATGGTGGATG 3'
14F： 5' ACACCCACCTCTGCTTCGT 3'
14R： 5' TGCCTTCACTCAAACCCTTT 3'
15F： 5' TGGGTGAAGAGCAAGGGTG 3'
15R： 5' GAGTGGGTGCAGTTGATGG 3'
166F：5' CCAGGAGCATGGCGAAAA 3'
16R： 5' AAGAGCCCCAGAAAGACCC 3'
17F：5' CTCACTTTCCAGAACCTT 3'
17R：5' TTATTTGACTCGACCTAA 3'
18F：5' CCTTCATTGCCCTTCACTC 3'
18R：5' CCTAGCCATCTTTCCTTGC 3'
19F：5' ATGTCTTCAAAGTTCTGGTG 3'
19R：5' AGATAGTGCATCCTGGGA 3'
20F：5' GGGTGGTGAAGGATGTTTG 3'
20R：5' TAGTCTAGGTTTGCGGGAGT 3'
21F：5' GCCCTATGGCTGCCTCTTA 3'
21R：5' CTCTGCTCCTTGGTCCTTCAC 3'
22F：5' TATGCCCTATGGCTGCCTCT 3'
22R：5' TGGTCCTTCACCTAACCTTGC 3'
23F：5' GGTCTGTCTCCTGGCATCAC 3'
23R：5' ACATCCTGGTCCCCTTTCA 3'
24F：5' ACAGGCACCGCAGCTCAT 3'
24R：5' CGCCACTCTGGGGACAAG 3'
25F：5' TCTCCACTGGCACCCTCC 3'
25R：5' GACGACCCCATTCTTCCCT 3'
26F：5' CTTCAAAGGGACACCTACGG 3'
26R：5' CAAAGCCTGGATACTGACACC 3'；
Again according to 1 pair of kinetochore primer sequence of kinetochore STR sequences Design:
27F：5' GGCACGGTGGCTCACGCCTGTAAT 3'
27R：5' GGGAGGCTAAGTTCCTTGTTCG 3'；
Wherein 1,2,3 ... 27 is the primer title, and F is upstream primer, and R is downstream primer.Further, 5 ' end of above-mentioned 27 pairs of primer sets is all added to universal tag sequence: CGCTTTACTGACGCGTTGC, for the secondary amplification of PCR product.
Her2 gene BAC clones structure, and its step is as follows:
1. genomic dna preparation: adopt commercially available DNA extraction test kit to extract the DNA in breast cancer tissue;
2. the genomic dna enzyme is cut: selectional restriction restriction endonuclease Hind III is carried out enzyme to genomic dna and is cut, and enzyme is cut product and carried out the DNA electrophoresis, after electrophoresis, chooses and reclaim the DNA fragmentation of 200～300kb fragment;
3. large fragment DNA and BAC clone is connected: adopting commercially available pIndigoBAC-5 is carrier, is 1:10 ratio structure linked system according to the mass ratio of carrier and large fragment DNA:
16 ℃, connect 16 hours; Process 20 minutes deactivation T4 ligase enzymes for 65 ℃; Connect product and dialysed 3 hours with the 0.025umMillipore film, the connection product of having dialysed is sub-packed in the centrifuge tube of 0.5ml, is transformed in the DH10B cell and cultivates screening;
4. Her2 gene clone screening: for Her2 gene design primer, the employing PCR method is screened the BAC clone of above-mentioned structure, the fragment that will have the PCR product reclaims, after proceeding to the T carrier, carry out sequencing analysis, comparison Her2 gene order, be with Her2 gene matcher the BAC clone who contains the Her2 gene.
Kinetochore STR sequence plasmid builds, and its step is as follows:
1. due to kinetochore STR sequence shorter (> 700bp), adopt the synthetic mode of chemical industry to obtain the STR sequence;
2. the STR sequence is connected with commercially available plasmid: adopting commercially available T vector plasmid is carrier, with 10ul system construction linked system:
3. after above-mentioned mixed solution light shaking, 12000rpm, 30 seconds are centrifugal, then are placed in 14 ℃ of incubated overnight (12-16h);
4. the product after connecting is put standby or-85 ℃ of prolonged preservation of 4 ℃ of refrigerators.
The Her2 gene BAC clone who primer sets pcr amplification embodiment 2 is prepared with 1-26 synthetic in embodiment 1, the kinetochore STR sequence plasmid prepared with the 27th couple of primer sets pcr amplification embodiment 3 synthesized in embodiment 1.
The concrete operation step of pcr amplification is:
1. cultivate BAC clone bacterium or STR sequence plasmid bacterium, be applied on culture medium flat plate, cultivate, select mono-clonal, shake-flask culture should be cloned bacterium, from the clone bacterium, extracting BAC cloned DNA or plasmid DNA;
2. with PCR reaction amplification, do not contain BAC clone or the plasmid DNA of tumor-necrosis factor glycoproteins, the PCR reaction system is 25ul:
The pcr amplification program:
3. to the electrophoretic analysis of carrying out of pcr amplification product, the results are shown in Figure 2.
As shown in Figure 2, the 1-26 group is the pcr amplification product electrophoresis result of Her2 gene, and 27 groups is the pcr amplification product electrophoresis result of kinetochore STR sequence, and all PCR product length is that 350-800bp does not wait.
In commercially available T carrier, in order to preserve, the PCR product cloning, in the T carrier, comprises the steps: by embodiment 4 gained PCR product cloning
1. get 4 sterilizing 200ul Eppendorf tubes, reaction system 10ul:
2. after above-mentioned mixed solution light shaking, 12000rpm, 30 seconds are centrifugal, then are placed in 14 ℃ of incubated overnight (12-16h);
3. the product after connecting is put standby or-85 ℃ of prolonged preservation of 4 ℃ of refrigerators.
When the needs secondary PCR increases, GCTTTACTGACGCGTTGC increases to the universal tag sequence C, upstream primer is CGCTTTACTGACGCGTTGC, downstream primer is GCAACGCGTCAGTAAGCG, amplification reaction system and response procedures are as described in Example 4, obtain a large amount of PCR products that does not contain tumor-necrosis factor glycoproteins, reduce production link and cost, enhance productivity.
A kind of Her2 gene does not contain the preparation of tumor-necrosis factor glycoproteins fluorescent probe, in the present embodiment, utilizes the PCR product prepared with 27 pairs of Auele Specific Primers in above-described embodiment 4, prepares the FISH probe by conventional random primer method.Use the random primer labelling test kit of Life Technology company, wherein use red fluorescence element labelling kit to prepare Her2 red fluorescence probe, use the green fluorescein labelling kit to prepare kinetochore green fluorescence probe.Fluorescently-labeled specific operation process is:
(1) 100mg PCR product D NA is dissolved in 24ul water, ice bath, add 20ul random primer solution, and mixed DNA boiled sex change 5 minutes, was placed in immediately on ice;
(2) add 5ul10 * dNTP solution, mix, then add 1ul Taq enzyme solution;
(3) after mixing, reactant was cultivated 1 hour in 37 ℃, is got 2ul and do the gel electrophoresis inspection, if the DNA probe fragment be labeled between 150-350bp, the mark best results;
10 minutes termination reactions of (4) 75 ℃ of heating, or add the 5ul reaction terminating liquid, namely obtain fluorescently-labeled FISH probe.
A kind of tumor-necrosis factor glycoproteins Her2 fluorescence probe in situ hybridization test kit that do not contain, comprise the double-colored probe of fluorescent in situ: Her2 red fluorescence probe and kinetochore green fluorescence probe, described double-colored probe is liquid reagent, by Her2 red fluorescence probe and kinetochore green fluorescence probe solution, obtain in the 200ul damping fluid, wherein Her2 red fluorescence concentration and probe concentration is 5ng/ul, kinetochore green fluorescence concentration and probe concentration is 5ng/ul, described damping fluid is: sodium-chlor 8.8g, Trisodium Citrate 4.4g, the 2x sodium citrate buffer solution that deionized water 400ml configuration forms.
This test kit also comprises: 4', 6-diamidino-2-phenylindone (DAPI) is redyed liquid 300ul.
Utilize the above-mentioned method that tumor-necrosis factor glycoproteins Her2 fluorescence probe in situ hybridization test kit detects the Her2 gene that do not contain:
1. the pre-treatment of testing sample paraffin section: a. through 10% formalin fix, paraffin-embedded tissue slice 3～5um, be placed on clean gluing slide, tissue slice is placed under 65 ℃ and toasts spend the night (12～16 hours), aging slide, again tissue slice is dipped in dimethylbenzene to room temperature dewaxing 2 times, each 10 minutes, immerse subsequently 100%(vt%) in ethanol 5 minutes; B. tissue slice is placed in to 100%(vt% successively) ethanol, 85%(vt%) ethanol and 70%(vt%) ethanol each 2 minutes, then tissue slice was at room temperature immersed in deionized water 3 minutes, after taking-up, draw unnecessary moisture with thieving paper; C. use 30%(wt%) acid S-WAT processed tissue slice 20～30 minutes under 50 ℃, under 90 ℃, cut into slices 30 minutes with water treatment, then under room temperature with rinsing in 2 * sodium citrate buffer solution 2 times, each 5 minutes, again tissue slice is soaked in 200ug/ul Proteinase K solution, 37 ℃, 10～20 minutes, use subsequently the rinsing of 2 * sodium citrate buffer solution 2 times, each 5 minutes; D. under room temperature, fix 5 minutes with formaldehyde, then tissue slice be placed in to 70%(vt% successively) ethanol, 85%(vt%) ethanol and 100%(vt%) ethanol dewaters, and each 2 minutes, finally, after the fragmentation seasoning, be heated to 56 degrees centigrade, standby;
2. probe and the nucleotide sequence hybridization of organizing to be measured: under a. room temperature, following liquid is joined in test tube,
Centrifugal 1～3 second, mix rear recentrifuge 1～3 second, test tube is placed in to 78 ℃ of water bath sex change 5 minutes, be placed in rapidly 45 ℃ of water baths, before hybridization, take out; B. above-mentioned pretreated slide is immersed to the 70%(wt% of 78 ℃ of preheatings) in methane amide/2 * sodium citrate buffer solution 30 minutes, be placed in successively again the 70%(vt% of-20 ℃ of precoolings) ethanol, 85%(vt%) ethanol and 100%(vt%) ethanol, each 2 minutes, seasoning under room temperature, be placed in 45 ℃ of preheatings 5 minutes; C. by the probe mixing drop of above-mentioned 10ul in fragmentation hybridization zone, add a cover immediately cover glass, use the rubber adhesive edge, slide is placed in to the box that wets, the hybridization of spending the night in 45 ℃;
3. slide washing: remove cover glass, slide is placed in to 0.3%(wt%) Nonidet P40/0.4 * sodium citrate buffer solution, vibrated 1～3 second, rinsing 2 minutes; Again fragmentation is placed in to 0.1%(wt%) Nonidet P40/2 * sodium citrate buffer solution, vibrated 1～3 second, rinsing 30 seconds; Finally slide is placed in to 70%(vt%) ethanol, rinsing 3 minutes;
4. redye: dark place is dried slide naturally, and 15ul DAPI counterstain is added on to hybridization zone, covered immediately, and placed 10～20 minutes dark place;
5. the FISH result is observed: use fluorescent microscope to observe hybridizing regional sample.
As shown in Figure 3, Fig. 3 is not for containing tumor-necrosis factor glycoproteins Her2 fluorescence probe in situ hybridization figure for result, and wherein template is the mammary cancer sample.It is 1. wherein kinetochore green fluorescence probe mark DNA fragmentation; 2. be Her2 red fluorescence labeled dna fragment; 3. be the red probe mark DNA fragmentation of Her2, show as the gene form that increases in a large number.In Fig. 3, A is that the Her2 gene is normal, and the quantity ratio of the DNA of the DNA of centromeric probe mark and Her2 probe mark is normal; B is the amplification of Her2 gene unconventionality, 1. with Her2 probe mark DNA fragmentation ratio 3., can find out that Her2 increases in a large number from green centromeric probe labeled dna fragment shown in white arrow figure.
Obviously, above-described embodiment is only for the example of doing clearly is described, and is not the restriction to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without also giving all embodiments.And within the apparent variation of therefore amplifying or change still be in the protection domain of the invention.
Claims (10)
1. FISH probe that does not contain tumor-necrosis factor glycoproteins for detection of the Her2 gene, it is characterized in that, described FISH DNA probe be take Her2 gene and centromeric sequence and is template, the nucleotide sequence that utilizes 27 pairs of PCR primer sets or have identity with primer sets, carrying out pcr amplification reaction prepares, wherein 1～26 pair of primer sets is Her2 gene PCR primer sets, and the 27th pair of primer sets is STR sequence pcr primer thing group, and described 27 pairs of primer sets are:
1 F：5' GGTTGGAATGAGTAAGAAGTAGC 3'
1 R：5' TAGGAATTTTCTAAAGGGGAGAC 3'
2 F：5' AGGTTAATGGTGGAAGTGGG 3'
2 R：5' ACACTCTACTATTAACATATGC 3'
3 F：5' TTTACTTTGGCTGCTGTTGC 3'
3 R：5' ATATTTTCGGATTTGGTATAGG 3'
4 F：5' TGGCTGTAGGTCAGAGTGGC 3'
4 R：5' AAGGATAAGGTGGAGTTTTGT 3'
5 F：5' TGGGTGATGGAATGATCTGG 3'
5 R：5' ACGAGTCTCCGAACAAAAGG 3'
6 F：5' AAAAGAAAACCAATGGGACAG 3'
6 R：5' CCCACGCCAACATAAATACAC 3'
7 F：5' TCCTCCCTCTTTGTCCTTATC 3'
7 R：5' AAAGTGCCCAGTTGAACTTGTT 3'
8 F：5' CTTTGTGGCAGCAGTTTCGT 3'
8 R：5' AGAGCAGATGGGTCAGAGGAC 3'
9 F：5' GTGGTGGGACTCAAAGACGG 3'
9 R：5' AGGAAATGGGTGGTGTAGGC 3'
10F：5' CCCTAGAAGGTGATGCTGATG 3'
10R：5' ATATGCTCCCATTTACAGAAAC 3'
11F：5' CCAGCCCACCCTGTCCTAT 3'
11R：5' TGCCCACTCTTACCCATCAA 3'
12F： 5' CCAGCCCACCCTGTCCTA 3'
12R： 5' TTCTGTCTCCTGCCATCCC 3'
13F： 5' TGCCTGACCTCAGCGTCTT 3'
13R： 5' GGTGGGTGTTATGGTGGATG 3'
14F： 5' ACACCCACCTCTGCTTCGT 3'
14R： 5' TGCCTTCACTCAAACCCTTT 3'
15F： 5' TGGGTGAAGAGCAAGGGTG 3'
15R： 5' GAGTGGGTGCAGTTGATGG 3'
166F：5' CCAGGAGCATGGCGAAAA 3'
16R： 5' AAGAGCCCCAGAAAGACCC 3'
17F： 5' CTCACTTTCCAGAACCTT 3'
17R： 5' TTATTTGACTCGACCTAA 3'
18F： 5' CCTTCATTGCCCTTCACTC 3'
18R： 5' CCTAGCCATCTTTCCTTGC 3'
19F： 5' ATGTCTTCAAAGTTCTGGTG 3'
19R： 5' AGATAGTGCATCCTGGGA 3'
20F： 5' GGGTGGTGAAGGATGTTTG 3'
20R： 5' TAGTCTAGGTTTGCGGGAGT 3'
21F： 5' GCCCTATGGCTGCCTCTTA 3'
21R： 5' CTCTGCTCCTTGGTCCTTCAC 3'
22F： 5' TATGCCCTATGGCTGCCTCT 3'
22R： 5' TGGTCCTTCACCTAACCTTGC 3'
23F： 5' GGTCTGTCTCCTGGCATCAC 3'
23R： 5' ACATCCTGGTCCCCTTTCA 3'
24F： 5' ACAGGCACCGCAGCTCAT 3'
24R： 5' CGCCACTCTGGGGACAAG 3'
25F： 5' TCTCCACTGGCACCCTCC 3'
25R： 5' GACGACCCCATTCTTCCCT 3'
26F： 5' CTTCAAAGGGACACCTACGG 3'
26R：5' CAAAGCCTGGATACTGACACC 3'；
27F：5' GGCACGGTGGCTCACGCCTGTAAT 3'
27R：5' GGGAGGCTAAGTTCCTTGTTCG 3'。
2. FISH probe according to claim 1, is characterized in that all inserting universal tag sequence: CGCTTTACTGACGCGTTGC at 5 ' end of described 27 pairs of Her2 gene PCR primer sets.
3. one kind comprises the described test kit that does not contain the FISH probe of tumor-necrosis factor glycoproteins for detection of the Her2 gene of claim 1～2.
4. test kit according to claim 3, it is characterized in that described FISH probe is the double-colored probe of fluorescence in situ hybridization, the double-colored probe of described FISH is pulvis, the double-colored probe of described FISH comprises Her2 red fluorescence probe and kinetochore green fluorescence probe, and the mass ratio of described Her2 red fluorescence probe and kinetochore green fluorescence probe is 1:1.
5. test kit according to claim 3, it is characterized in that described FISH probe is the double-colored probe of fluorescence in situ hybridization, the double-colored probe of described FISH is liquid reagent, the double-colored probe of described FISH is to obtain by Her2 red fluorescence probe and kinetochore green fluorescence probe are dissolved in to the 2x sodium citrate buffer solution, wherein the concentration of Her2 red fluorescence probe is 5ng/ul(w/v), the concentration of kinetochore green fluorescence probe is 5ng/ul(w/v).
6. test kit according to claim 3, is characterized in that described test kit also comprises 4', and 6-diamidino-2-phenylindone (DAPI) is redyed liquid.
7. one kind is utilized the described test kit of claim 3～6 to the method that the Her2 gene detects, and it is characterized in that, comprises the steps: that (1) is fixed on tissue to be detected on slide, and the tissue to be measured on slide is carried out to pre-treatment; (2) tissue to be measured on probe and slide is carried out to the sex change hybridization of nucleotide sequence; (3) after hybridization, slide is washed; (4) use and to redye liquid the hybridization zone on slide is redyed; (5) use the hybridization zone of fluorescence microscope slide.
8. detection method according to claim 7, the nucleotide sequence sex change hybridization that it is characterized in that the described probe of step (2) and tissue to be measured is: (1) gets each 1ul of probe reagent, 2x sodium citrate buffer solution 7ul, deionized water 1ul, be mixed to get the probe mixed solution, sex change is 5 minutes under 78 ℃ of conditions, then is placed in rapidly under 45 ℃ of conditions, takes out before hybridization; (2) slide of organizing to be measured after pretreatment was immersed in 78 ℃ of methane amide/2 * sodium citrate buffer solutions to denaturing treatment 30 minutes, be placed in successively again the 70%(vt% of-20 ℃ of precoolings), 85%(vt%), 100%(vt%) the ethanol gradient solution carries out processed, then by after the slide seasoning, be placed in 45 ℃ of condition preheatings 5 minutes; (3) the probe mixing drop after above-mentioned sex change is regional in slide hybridization, be placed under 45 ℃ of conditions and hybridized 12～16 hours.
9. detection method according to claim 7, the pre-treatment that it is characterized in that the described tissue to be detected of step (1) is: (1) is placed in paraffin organization section to be measured on fragmentation, slide is placed under 65 ℃ and toasted 12～16 hours again, then with dimethylbenzene dewaxing, subsequently slide is placed in to 100%(vt% successively), 85%(vt%), 70%(vt%) the ethanol gradient solution carries out hydration process; (2) with acid S-WAT, process and organized slide 20～30 minutes under 50 ℃, under 90 ℃, use water treatment slide 30 minutes, use again the rinsing of 2 * sodium citrate buffer solution, then slide is immersed in Proteinase K solution to 10～20 minutes under 37 ℃, use subsequently the rinsing of 2 * sodium citrate buffer solution; (3) with the fixing slide of formaldehyde, then slide is placed in to the ethanol gradient solution dewaters, finally by naturally drying, and be heated to 56 ℃, standby.
10. detection method according to claim 7, is characterized in that the described slide washing of step (3) is: slide is placed in to 0.3%(wt%) Nonidet P40/0.4 * sodium citrate buffer solution, rinsing 2 minutes; Again fragmentation is placed in to 0.1%(wt%) Nonidet P40/2 * sodium citrate buffer solution, rinsing 30 seconds; Finally slide is placed in to 70%(vt%) ethanol, rinsing 3 minutes.
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