WO2017114010A1 - Sonde de détection du gène top2a, son procédé de préparation et kit d'essai - Google Patents

Sonde de détection du gène top2a, son procédé de préparation et kit d'essai Download PDF

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WO2017114010A1
WO2017114010A1 PCT/CN2016/105717 CN2016105717W WO2017114010A1 WO 2017114010 A1 WO2017114010 A1 WO 2017114010A1 CN 2016105717 W CN2016105717 W CN 2016105717W WO 2017114010 A1 WO2017114010 A1 WO 2017114010A1
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probe
top2a gene
top2a
ctd
gene detecting
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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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Guangzhou LBP Medicine Science and Technology Co Ltd
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • 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
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Definitions

  • the invention belongs to the biotechnology, in particular to a TOP2A gene detection probe and a preparation method and kit thereof.
  • the TOP2A gene is a gene encoding DNA topoisomerase II alpha (TOPII ⁇ ), the biological role of DNA topoisomerase, and the first is to regulate the supercoiled state of DNA and the ligated or unknotted DNA. State, which indirectly affects the process of nucleic acid metabolism in cells; second, it is directly involved in the process of cellular processes, DNA recombination, repair, transcription and replication that need to be interrupted and reattached. Patients with abnormal TOP2A gene have poor prognosis and shortened recurrence-free survival, especially in patients with TOP2A gene deletion. Gene amplification suggests a recurrence of the tumor, or a long-term decline in efficacy.
  • TOPII ⁇ DNA topoisomerase II alpha
  • Anthracyclines are the cornerstone of postoperative adjuvant chemotherapy for breast cancer, and studies have confirmed that TOP2A gene is associated with anthracycline efficacy [Brase, JC, Schmidt, M, Fischbach, T, Sultmann, H, Bojar, H, Koelbl, H (2010) ERBB2and TOP2A in breast cancer: a comprehensive analysis of gene amplification, RNA levels, and protein expression and their influence on prognosis and prediction.
  • the TOP2A gene is located in the 17q21 region of chromosome 17, and the abnormalities of TOP2A are classified into two cases of amplification and deletion. FISH detection can make a clear judgment for these two 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. Strong hybridization signal, easy to detect; 2) whole chromosome or chromosomal region-specific probe consisting of a very different nucleotide fragment on a chromosome or a segment of a chromosome, which can be cloned into phage and plasmid A chromosome-specific large fragment is obtained; 3) a specific position probe consisting of one or several cloned sequences.
  • the fluorescein labeling of the probe can be performed by direct and indirect labeling.
  • Indirect labeling is performed by biotin-labeled DNA probes, which are detected by fluorescein avidin or streptavidin after hybridization.
  • the avidin-biotin-fluorescein complex can be used to amplify the fluorescent signal, 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 TOP2A gene detecting probe and a preparation method thereof, and the prepared probe can be used for detecting the TOP2A gene state, that is, detecting the finger copy number change of the TOP2A gene, and has good specificity.
  • a method for preparing a TOP2A gene detection probe comprises the following steps:
  • a BAC clone as at least one of RP11-1152A10 and CTD-3217C5, or selecting a BAC clone as at least one of RP11-737D6, CTD-3087O22, RP11-48O10, and CTD-2134K5;
  • the plasmid DNA is labeled with fluorescein, and the fluorescein labeled by the plasmid DNA of different sources is the same, that is, it is obtained.
  • the BAC clones are RP11-1152A10 and CTD-3217C5.
  • the BAC clones are RP11-737D6, CTD-3087O22, RP11-48O10, and CTD-2134K5.
  • 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 TOP2A gene detecting kit.
  • a TOP2A gene detection kit comprising the above 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 TOP2A gene detection probe.
  • the present invention detects the TOP2A gene copy number by FISH (Fluorescence In-Situ Hybridization) method by screening the optimal TOP2A gene detection probe and its combination.
  • 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.
  • the lack of clinical detection of TOP2A mutation is beneficial to screening more patients who benefit from targeted drugs and improving the survival rate and overall survival of breast cancer patients.
  • the TOP2A state change is known from the gene level, various The signal type shows the genetic diversity of tumor cells in solid tissues, which can be applied in the fields of tumor biology and cytogenetics. It can help comprehensively evaluate each molecular marker and assist in the selection of clinical targeted therapy drugs and treatment options for breast cancer.
  • Figure 1 is a schematic illustration of the detection probe sequence in Example 1.
  • Fig. 2 is a graph showing the results of FISH detection of human peripheral blood cultured cell sheets in Example 1.
  • Fig. 3 is a graph showing the results of FISH detection of breast cancer tissue samples in Example 4, wherein the detection signal type is 2R2G, and the TOP2A gene is not amplified.
  • Example 4 is a diagram showing the results of FISH detection of breast cancer tissue samples in Example 4, wherein the detection signal type is 6-12R2G, and the TOP2A gene is amplified.
  • the preparation method of the TOP2A detection probe of the present embodiment includes the following steps.
  • GSP TOP2A includes two groups, including a first probe, a second probe, a third probe, and a fourth probe, respectively. Specifically, the following table is purchased from the Invitrogen RP11 BAC 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
  • Probe set 1 BAC Insert start and end position First probe RP11-1152A10 Chr17:38376940...38546337(169Kb) Second probe CTD-3217C5 Chr17:38521566...38730400(209Kb)
  • the plasmid DNA mixture was fluorescently labeled by a nick translation method, and each probe labeled fluorescein was Spectrum-Orange. Using abbott's Nick Translation Kit, strictly as protected from light The PCR reaction system was prepared on ice under the conditions.
  • 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:
  • chromosome 17 discriminating probe can be labeled with chromosome 17, purchased from SE17, D17Z1 (KBI-20017, KREATECH) shows green fluorescence.
  • the TOP2A gene probe signal is bright, and the sensitivity and specificity of 100% can be observed on the metaphase chromosome in human peripheral blood cultured cell sheets; the TOP2A gene copy number can be clearly recorded by using the paraffin sample piece for hybridization detection.
  • the TOP2A gene detection kit includes two components of a TOP2A hybridization solution and a DAPI counterstaining agent, wherein the TOP2A hybridization solution comprises the GSP TOP2A gene probe described in Example 1 (two sets of detection probes respectively, corresponding to two kits) ), CSP17 probe (Chromosome 17 probe), buffer component for hybridization environment (promoting hybridization), COT Human DNA for blocking repeats, and the like.
  • 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 CSP17 probe shows a green signal
  • the GSP TOP2A probe is a red signal.
  • Example 2 Using the two sets of detection probes described in Example 1, the test kit described in Example 2 was tested on 20 clinical samples (which were confirmed by pathological examination, see the table below). According to the detection method of Example 3, the detection was repeated 3 times, the results were consistent, and the repeatability of the detection results was good; the detection consistency of the two probe combinations was good. Compared with commercially available commercial reagents, the test results are completely consistent, and the specificity and sensitivity of the reagents are high.
  • 3 and 4 are graphs showing the results of the probe set 1. The results of probe set 2 were the same as those of probe set 1, and the figures are omitted.
  • Figure 3 shows the results of the negative sample test. The typical signal type is 2R2G. The result is that the TOP2A gene is not amplified.
  • Figure 4 shows the positive sample detection result.
  • the signal type is 2 ⁇ 6R/2G.
  • the result is judged as TOP2A gene amplification.
  • the red signal in the figure shows GSP TOP2A, and the green signal shows CSP 17 (used to locate the centromere probe on chromosome 17).
  • the detection can also be achieved by using one of the TOP2A genes, respectively, and the specific results are omitted.
  • the use of the combined probes and the detection signal are better with respect to the combination of the probes. Theoretically, the longer the length of the probe, the brighter the fluorescence signal obtained during actual detection, but because more gene sequences may be involved, the complexity of the resulting signal is increased, and the difficulty of detection is also enhanced.
  • the total lengths of the BAC clones of the detection probes of Group 1 and Group 2 for 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.

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Abstract

L'invention concerne une sonde de détection de gène TOP2A et son procédé de préparation, le procédé comprenant les étapes suivantes : sélectionner un clone BAC pour qu'il soit au moins un clone parmi RP11-1152A10 et CTD-3217C5, ou sélectionner le clone BAC pour qu'il soit au moins un clone parmi RP11-737 D6, CTD -3087O22, RP11-48O10 et CTD-2134 K5 ; effectuer respectivement effectuer une extraction plasmidique sur les clones, obtenir l'ADN plasmidique et quantifier ; marquer à la fluorescéine. L'invention concerne également un kit de test comprenant la sonde de détection du gène TOP2A.
PCT/CN2016/105717 2015-12-30 2016-11-14 Sonde de détection du gène top2a, son procédé de préparation et kit d'essai Ceased WO2017114010A1 (fr)

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CN105483255A (zh) * 2015-12-30 2016-04-13 广州安必平医药科技股份有限公司 Top2a基因检测探针及其制备方法和试剂盒
CN105524990A (zh) * 2015-12-30 2016-04-27 广州安必平医药科技股份有限公司 Her-2基因和/或top2a基因检测探针及其制备方法和试剂盒

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CN102399772A (zh) * 2010-09-15 2012-04-04 中山大学达安基因股份有限公司 乳腺癌分子标志物相关探针的制备方法及其应用
CN105483255A (zh) * 2015-12-30 2016-04-13 广州安必平医药科技股份有限公司 Top2a基因检测探针及其制备方法和试剂盒

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AU2009250169A1 (en) * 2008-05-20 2009-11-26 Dako Denmark A/S A combined method for predicting the response to an anti-cancer therapy
CN104561360A (zh) * 2015-01-30 2015-04-29 益善生物技术股份有限公司 Top2a基因异常检测探针、试剂盒以及方法

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CN102399772A (zh) * 2010-09-15 2012-04-04 中山大学达安基因股份有限公司 乳腺癌分子标志物相关探针的制备方法及其应用
CN105483255A (zh) * 2015-12-30 2016-04-13 广州安必平医药科技股份有限公司 Top2a基因检测探针及其制备方法和试剂盒

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KOWALSKA: "A new platform linking chromosomal and sequence information", CHROMOSOME RESEARCH, vol. 15, 10 May 2007 (2007-05-10), pages 3, XP019499043 *

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