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WO2017114011A1 - Sonde de détection de gène her-2 et/ou de gène top2a, son procédé de préparation, et kit d'essai - Google Patents

Sonde de détection de gène her-2 et/ou de gène top2a, son procédé de préparation, et kit d'essai Download PDF

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WO2017114011A1
WO2017114011A1 PCT/CN2016/105718 CN2016105718W WO2017114011A1 WO 2017114011 A1 WO2017114011 A1 WO 2017114011A1 CN 2016105718 W CN2016105718 W CN 2016105718W WO 2017114011 A1 WO2017114011 A1 WO 2017114011A1
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gene
top2a
ctd
probe
bac
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陈绍宇
何瑰
席影
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Guangzhou LBP Medicine Science and Technology Co Ltd
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Definitions

  • the present invention belongs to the field of biotechnology, and particularly relates to a HER-2 (ERBB2) gene and/or TOP2A gene detecting probe, a preparation method thereof and a kit.
  • HER-2 ERBB2
  • TOP2A TOP2A gene detecting probe
  • HER-2 The HER-2 (ERBB2) gene has gene amplification and protein overexpression in 20%-30% of breast cancer patients.
  • HER-2 is currently recognized as an important prognostic/predictor for breast cancer.
  • HER-2 is an independent prognostic factor for tumor recurrence and overall survival.
  • HER-2 positive breast cancer is invasive, with short disease-free survival and poor prognosis.
  • Trastuzumab (Herceptin) is indicated for breast cancer with HER-2 overexpression and gene amplification.
  • TOP2A gene regulates the supercoiled state of DNA, directly participates in cellular processes, and is a target enzyme of anthracycline antibiotics.
  • the HER-2 gene is located in the 17q12 region of chromosome 17, the abnormality of HER-2 is amplified; the TOP2A gene is located in the 17q21 region of chromosome 17, and the abnormality of TOP2A is divided into amplification and deletion, and FISH detection can be used for this. A clear judgment is made in both cases.
  • Fluorescence in situ hybridization is a non-radioactive in situ hybridization technique developed on the basis of the original radioactive in situ hybridization technique in the late 1980s. At present, this technology has been widely used in animal and plant genomic structure research, chromosome fine structure variation analysis, viral infection analysis, human prenatal diagnosis, tumor genetics and genome evolution research in many fields.
  • the basic principle of FISH is to use a known labeled nucleic acid as a probe to heterologously bind to an unknown single-stranded nucleic acid in a material to be tested according to the principle of base complementation to form a hybrid double-stranded nucleic acid which can be detected.
  • the probe can be directly hybridized to the chromosome to localize the specific gene on the chromosome.
  • fluorescence in situ hybridization has the characteristics of rapid detection signal, high hybridization specificity and multi-staining, so it has received widespread attention in the field of molecular cytogenetics.
  • the probes used for hybridization can be roughly classified into three categories: 1) chromosome-specific repeat probes, such as alpha satellites, satellite class III probes, which often have a hybrid target of more than 1 Mb, do not contain scattered repeats, and bind tightly to the target. , hybrid signal is strong, easy to detect; 2) whole chromosome or chromosomal region-specific probe, which is on a chromosome or a segment on a chromosome Consisting of extremely different nucleotide fragments, which can be obtained from chromosome-specific large fragments cloned into phage and plasmids; 3) specific position probes consisting of one or several cloned sequences.
  • the fluorescein labeling of the probe can be performed by direct and indirect labeling.
  • the indirect labeling is a biotin-labeled DNA probe, which is detected by fluorescein avidin or streptavidin after hybridization, and the avidin-biotin-fluorescein complex can also be used to fluoresce signals. Amplification is performed so that a fragment of about 500 bp can be detected.
  • the direct labeling method is to directly bind fluorescein to the probe nucleotide or the pentose phosphate backbone, or to incorporate fluorescein nucleoside triphosphate in the nick translation labeling probe.
  • the direct labeling method has simple steps in detection and is convenient for clinical use.
  • One of the objects of the present invention is to provide a HER-2 gene and/or TOP2A gene detecting probe and a preparation method thereof, which can be used for detecting the HER-2 gene and the TOP2A gene state, that is, detecting the HER-2 gene. And the change in the copy number of the TOP2A gene has good specificity.
  • a method for preparing a HER-2 gene and/or a TOP2A gene detection probe comprising the steps of:
  • the BAC clone targeting the HER-2 gene is at least one of RP11-1021P11, RP11-62N23, and RP11-1044P23, or the BAC clone is at least one of RP11-98J2, RP11-1065L22, and CTD-2327I15. And/or selecting a BAC clone for the TOP2A gene as at least one of RP11-1152A10, CTD-3217C5, or selecting a BAC clone as at least one of RP11-737D6, CTD-3087O22, RP11-48O10, CTD-2134K5;
  • the plasmid DNA is labeled with fluorescein, and the fluorescein labeled with the plasmid DNA of the same gene is the same, and the color of the fluorescein labeled with the HER-2 gene and the detection probe for the TOP2A gene is different.
  • the BAC clones for the TOP2A gene are RP11-1152A10 and CTD-3217C5.
  • the BAC clones for the TOP2A gene are RP11-737D6, CTD-3087O22, RP11-48O10, and CTD-2134K5.
  • the BAC clones directed against the HER-2 gene are RP11-1021P11, RP11-62N23, and RP11-1044P23.
  • the BAC clones for the TOP2A gene are RP11-98J2, RP11-1065L22, and CTD-2327I15.
  • the labeled fluorescein selects a fluorescent dye known in the art, preferably fluorescein is Alexa FITC, Alexa Rhodamine, Texas Red, pacific DEAC.
  • the labeling of the gene probe can be performed by labeling the corresponding fluorescein to the double-stranded nucleic acid by methods in the prior art, including but not limited to: random primer method, nick translation, etc., marker gene probe
  • the needle can be a commercially available nick translation labeling kit and/or a random primer labeling kit, preferably abbott and/or Roche's Nick Translation Kit.
  • the plasmid DNA is preferably subjected to fluorescein labeling by a random primer method or a nick translation method.
  • the label has a temperature of from 24 ° C to 26 ° C and a marked time of from 4 to 6 hours.
  • Another object of the present invention is to provide a HER-2 gene and TOP2A gene detecting kit.
  • a HER-2 gene and/or TOP2A gene detection kit comprising the above HER-2 gene and/or TOP2A gene detection probe.
  • a chromosome 17 discrimination probe (CSP17) probe for internal control is included, the identification probe being different in color from the fluorescein labeled by the HER-2 gene and the TOP2A gene detection probe.
  • CSP17 chromosome 17 discrimination probe
  • the present invention uses the FISH (Fluorescence In-Situ Hybridization) method to detect the copy number of the HER-2 gene and the TOP2A gene by screening the optimal HER-2 gene and/or TOP2A gene detection probes and their combinations.
  • FISH Fluorescence In-Situ Hybridization
  • the preferred clone of the present invention has good detection specificity and high sensitivity.
  • the length probe is limited to about 500 bp, which improves the hybridization efficiency and reduces the hybridization background.
  • the signal counting line is accurate and fast, and the result is reproducible; through the simultaneous detection and evaluation of HER-2, TOP2A copy number and chromosome 17, it is beneficial to determine the treatment plan and screen more breasts that benefit from targeted drugs. Cancer patients improve patient survival and overall survival.
  • the HER-2 gene and TOP2A kit of the present invention the HER-2 gene and TOP2A state changes are known from the gene level, and various signal types exhibit tumor cell genetic diversity of solid tissues, which can be realized in tumor organisms.
  • the application in the fields of learning and cytogenetics can help comprehensively evaluate various molecular markers, assist breast cancer prognosis and clinical targeted therapy drugs and treatment options.
  • Figure 1A is a schematic illustration of the HER-2 gene detection probe sequence of Example 1.
  • Fig. 1B is a schematic diagram showing the TOP2A gene detecting probe sequence of Example 1.
  • Fig. 2 is a graph showing the results of FISH detection of the HER-2 gene and the TOP2A gene detecting probe of human peripheral blood culture cell sheets in Example 1.
  • Example 3 is a diagram showing the results of FISH detection of breast cancer tissue samples in Example 4, wherein the detection signal type is 2R2G2A, the HER-2 gene is not amplified, and the TOP2A gene is not amplified.
  • Example 4 is a diagram showing the results of FISH detection of a breast cancer tissue sample in Example 4, wherein the detection signal type is clustered R cluster G2A, and the HER-2 gene and the TOP2A gene are co-amplified.
  • a method for preparing a HER-2 detection probe comprising the following steps:
  • Cloning screening Clones containing the target gene HER-2 and the sequences at both ends were selected, as shown in Fig. 1A.
  • GSP HER-2 consists of two groups, and the following two sets of detection probes are prepared separately.
  • HER-2 chr17 37, 856, 254-37, 884, 915, 28, 662 bp
  • HER-2 probe set 1 BAC Insert start and end position First probe RP11-1021P11 Chr17:37497503...37725641(228Kb) Second probe RP11-62N23 Chr17:37725641...37882864(157Kb) Third probe RP11-1044P23 Chr17:37871783...38066611(195Kb) HER-2 probe set 2 BAC Insert start and end position Fourth probe RP11-98J2 Chr17:37572511...37753435 (180Kb) Fifth probe RP11-1065L22 Chr17:37758567...37990799(232Kb) Sixth probe CTD-2327I15 Chr17:37963139...38082538(119Kb)
  • the preparation method of the TOP2A detection probe comprises the following steps:
  • Cloning Screening Clones containing the TOP2A gene of interest and the sequences at both ends were selected, as shown in Figure 1B.
  • GSP TOP2A consists of two groups, as shown in the following table, which were purchased from the Invitrogen RP11BAC and CTD BAC clone libraries. The following two sets of detection probes were separately prepared.
  • TOP2A chr17 38, 544, 773-38, 574, 202, 29, 430 bp
  • the plasmid DNA mixture was fluorescently labeled by the nick translation method.
  • the fluorescein labeled for each probe of the GSP HER-2 gene was Spectrum-Orange, and the fluorescein labeled for each probe of the GSP TOP2A gene was Spectrum- Green.
  • the PCR reaction system was prepared on ice under strict light conditions as follows.
  • the labeled product was subjected to ethanol precipitation and concentration, and sodium acetate and absolute ethanol were sequentially added to a 1.5 ml centrifuge tube in the following manner, and protected from light and ice:
  • GSP HER-2 and GSP TOP2A gene probe validation hybridization solution prepared using HER-2 probe set 1 + TOP2A probe set 1 and HER-2 probe set 2+ TOP2A probe set 2, respectively
  • the hybridization solution is a reagent to be detected, and probe verification is performed using human normal division metaphase lymphocyte droplets (detection method is referred to in Example 3).
  • the cultured cells contain metaphase or interphase chromosomal DNA. When fluorescent in situ hybridization, the chromosomal DNA appears as a morphologically recognizable chromosome or nucleus.
  • Figure 2 (experimental results applied to the HER-2 probe set 1 + TOP2A probe set 1) shows the results of FISH hybridization of metaphase chromosomes.
  • the red signal shows GSP HER-2
  • the green signal shows GSP TOP2A
  • the cyan signal shows CSP17 (for positioning the chromosome 17 centromere probe, purchased from D17Z1SpectrumAqua Probe, article number: 06J38-017).
  • the HER-2/TOP2A/17 chromosome probe signal is bright, and sensitivity and specificity can be observed on the metaphase chromosome in human peripheral blood cultured cells. 100% can be clearly recorded by using paraffin sample tablets for hybridization detection.
  • Three target copy numbers The results of other corresponding probe verification experiments are the same as those of the HER-2 probe set 1 + TOP2A probe set 2, and the figures are omitted.
  • the HER-2 gene and TOP2A gene detection kit includes two components of HER-2/TOP2A hybridization solution and DAPI counterstaining agent, wherein the HER-2 and TOP2A hybridization liquids comprise the set of GSP TOP2A gene probes described in Example 1.
  • the HER-2 gene and TOP2A gene detection kits are of the following four types: HER- 2 (group 1) + TOP2A (group 1) combination, HER-2 (group 2) + TOP2A (group 2) combination, HER-2 (group 1) + TOP2A (group 2) combination, HER-2 ( Group 2) + TOP2A (Group 1) combination, CSP17 probe (Chromosome 17 identification Needle, purchased from D17Z1 SpectrumAqua Probe, item number: 06J38-017), buffer component for hybridization environment (promoting hybridization), closed repetitive sequence of COT Human DNA, etc.).
  • DAPI counterstaining agent is mainly used for counterstaining of cells after hybridization, in which DAPI binds to DNA, so that the nucleus shows blue fluorescence, and the counterstaining agent containing p-phenylenediamine can maintain fluorescence stability.
  • the reaction time of pepsin needs to be determined by preliminary tests. Samples prepared in the same batch can be pre-tested as described, usually at intervals of 5 minutes. For example, the digestion time is 5 minutes, 10 minutes, and 15 minutes, respectively. After the "slide pretreatment" is completed, the tissue digestion state can be observed under a bright field using a 10 ⁇ or 20 ⁇ objective lens; or DAPI counterstaining can be directly performed. The digestion state is judged.
  • the above listed reagents were all prepared in a circular dyeing tank (40 ml each), and up to 5 slices per dyeing tank. For non-room temperature solutions, pre-heat the reagents to the specified temperature before starting the operation. During the washing process, the dyeing tank can be gently shaken at intervals of 2 to 3 minutes to improve the washing effect.
  • the relevant fluorescence and DAPI need to be observed with a suitable filter block.
  • the red signal indicates GSP HER-2
  • the green signal indicates GSP TOP2A
  • the cyan signal indicates CSP 17.
  • GSP TOP2A (red) and CSP17 (green) signals were counted in five clear tumor regions, and the number of GSP TOP2A and CSP 17 signals in individual nuclei was counted.
  • Example 2 Using the combination of the HER-2 gene and the TOP2A gene detection probe described in Example 1, the combination of the four detection kits described in Example 2 (HER-2 (probe group 1) + TOP2A (probe group 1), HER -2 (probe group 2) + TOP2A (probe group 2) combination, HER-2 (probe group 1) + TOP2A (probe group 2) combination, HER-2 (probe group 2) + TOP2A (Combination of probe set 1)) For 20 clinical samples (which were confirmed by pathological examination, see the table below), respectively. The detection of the two probe combinations is consistent. Compared with commercially available commercial reagents, the test results are completely consistent, and the specificity and sensitivity of the reagents are high.
  • Figure 3 and Figure 4 show the results of the combined kit detection of HER-2 (probe group 2) + TOP2A (probe group 2), as shown in Figure 3, FISH detection results of paraffin-embedded tissue samples of breast cancer, signal type The expression was 2R2G2A. The results showed that there was no abnormality in the HER-2/TOP2A gene and the chromosome 17 was normal.
  • the FISH detection results of the breast cancer tissue samples showed that the signal type was clustered R clustered G2 ⁇ 6A, and the result was judged as The HER-2/TOP2A gene was co-amplified, and chromosome 17 was multiplied.
  • the results of the combination of the other two different gene probes were the same, and the figures were omitted.
  • the detection of the HER-2 gene and the TOP2A gene can be achieved by using one probe, respectively, but the detection signal can be better by using the combination probe in combination with the probe.
  • the BAC clones of the probe set 1 and probe set 2 of the HER-2 gene of the present invention have lengths of 510 Kb and 569 Kb, respectively, and are nucleic acid mixtures containing the HER-2 gene and its both ends. .
  • the total lengths of the BAC clones of the probe sets 1 and 2 of the TOP2A gene of the present invention are: 353 Kb and 576 Kb, respectively, which are nucleic acid mixtures comprising the TOP2A gene and its both ends.
  • the samples can be molecularly classified according to the detection results, and used for clinical treatment plan formulation, drug selection and efficacy judgment according to the significance of the detection indicators.

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Abstract

L'invention concerne une sonde de détection de gène HER-2 et/ou de gène TOP2A, et son procédé de préparation, le procédé comprenant les étapes suivantes : sélectionner un clone BAC pour que le gène HER-2 soit RP11-1021P11, RP11-62N23 ou RP11-1044P23 ou sélectionner le clone BAC pour qu'il soit RP11-98J2, RP11-1065L22 ou CTD-2327I15, et/ou sélectionner un clone BAC pour que le gène TOP2A soit RP11-1152A10 ou CTD-3217C5 ou sélectionner le clone BAC pour qu'il soit RP11-737D6, CTD-3087O22, RP11-48O10 ou CTD-2134K5 ; obtenir l'ADN plasmidique ; et procéder à son marquage. Un kit d'essai contenant la sonde de détection de gène HER-2 et/ou de gène TOP2A est en outre décrit. L'invention est précise et rapide en termes de rangées de comptage de signaux, et offre une bonne reproductibilité des résultats.
PCT/CN2016/105718 2015-12-30 2016-11-14 Sonde de détection de gène her-2 et/ou de gène top2a, son procédé de préparation, et kit d'essai Ceased WO2017114011A1 (fr)

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CN109609631B (zh) * 2018-12-19 2020-01-24 益善生物技术股份有限公司 一种top2a基因的fish探针的制备方法
CN110208056B (zh) * 2019-06-06 2021-08-31 厦门大学附属第一医院 胃癌her-2 fish的组织芯片制作方法

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