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WO2017114005A1 - Sonde de détection du gène terc et/ou du gène myc, son procédé de préparation, et trousse de réactifs - Google Patents

Sonde de détection du gène terc et/ou du gène myc, son procédé de préparation, et trousse de réactifs Download PDF

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WO2017114005A1
WO2017114005A1 PCT/CN2016/105711 CN2016105711W WO2017114005A1 WO 2017114005 A1 WO2017114005 A1 WO 2017114005A1 CN 2016105711 W CN2016105711 W CN 2016105711W WO 2017114005 A1 WO2017114005 A1 WO 2017114005A1
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gene
terc
myc
ctd
probe
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何瑰
陈绍宇
席影
张会清
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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
    • C12N15/10—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
    • 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
    • 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
    • C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation

Definitions

  • the present invention belongs to the field of biotechnology, and particularly relates to a TERC (ERBB2) gene and/or MYC gene detecting probe, a preparation method thereof and a kit.
  • TERC ERBB2
  • MYC MYC gene detecting probe
  • Cervical cancer is a common malignant tumor in gynecology. From normal cervix, cervical intraepithelial neoplasia to cervical cancer is a gradual development process, its occurrence and development are closely related to the abnormal expression or deletion of various genes and proteins, which is a complex multi-step process. During the treatment of cervical cancer, the therapeutic effect on cervical cancer is related to the pathological grade. CIN and early cervical cancer (including carcinoma in situ and early invasive carcinoma) have very good therapeutic effects, and the treatment measures are appropriate. survive. Therefore, early diagnosis and accurate grading of cervical cancer is very important in the prevention and treatment of cervical cancer. Human telomerase RNA component (TERC) is a major component of human telomerase.
  • TTC Human telomerase RNA component
  • the protooncogene MYC v-myc avian myelocytomatosis viral oncogene homolog, MYC
  • MYC v-myc avian myelocytomatosis viral oncogene homolog
  • 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 letter Strong, easy to detect; 2) Whole-chromosomal or chromosomal region-specific probes consisting of extremely different nucleotide segments on a chromosome or a segment of a chromosome, which can be cloned into phage and plasmid-specific chromosomes Large fragment acquisition; 3) specific position probe 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 TERC gene and/or MYC gene detecting probe and a preparation method thereof, which can be used for detecting the state of the TERC gene and the MYC gene, that is, detecting a copy of the TERC gene and the MYC gene.
  • the number changes and has good specificity.
  • a method for preparing a TERC gene and/or a MYC gene detection probe comprising the steps of:
  • a BAC clone for the TERC gene as at least one of CTD-3214J12, RP11-816J6, and RP11-778I3, or selecting a BAC clone as at least one of RP11-3K16, CTD-2582E13, and RP11-886J24; / or select the BAC clone for the MYC gene as CTD-2511O8, or select the BAC clone as at least one of RP11-440N18 and CTD-2205H22;
  • 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 fluoresin labeled with the TERC gene and the detection probe for the MYC gene is different.
  • the BAC clones for the TERC gene are CTD-3214J12, RP11-816J6, and RP11-778I3.
  • the BAC clones for the TERC gene are RP11-3K16, CTD-2582E13, and RP11-886J24.
  • the BAC clone for the MYC gene is CTD-2511O8.
  • the BAC clones directed against the MYC gene are RP11-440N18 and CTD-2205H22.
  • 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 14 ° C to 18 ° C and a labeling time of from 10 to 14 hours.
  • Another object of the present invention is to provide a TERC gene and MYC gene detecting kit.
  • a TERC gene and/or MYC gene detection kit comprising the above TERC gene and/or MYC gene detection probe.
  • a chromosome 7 discrimination probe (CSP 7) probe for internal control is included, the identification probe being different in color from the fluorescein labeled by the TERC gene and the MYC gene detection probe.
  • CSP 7 chromosome 7 discrimination probe
  • the present invention detects the TERC/MYC gene amplification by FISH (Fluorescence In-Situ Hybridization) by screening the optimal MYC gene amplification detection probe and its combination, and the signal counting is accurate and rapid, and The reproducibility of the results is good; supplementing the shortage of detection reagents in the clinic depends on the import, which is beneficial to the subsequent application of the detection probe in clinical research, early diagnosis, grading diagnosis and guiding treatment of cervical cancer.
  • FISH Fluorescence In-Situ Hybridization
  • the cloning detection specificity of the present invention is good, and the TERC/MYC gene amplification detection kit according to the present invention understands the state change of the MYC gene from the gene level, and changes the abnormal signal pattern and the number of abnormal signal cells. Judging pathological grades is diagnostic in cytological height and low-grade lesions.
  • Fig. 1A is a schematic diagram showing the TERC gene detecting probe sequence of Example 1.
  • Figure 1B is a schematic illustration of the MYC gene detection probe sequence of Example 1.
  • FIG. 2 is a FISH detection result of a human peripheral blood culture cell sheet TERC gene and a MYC gene detection probe in Example 1.
  • Example 3 is a FISH detection result of a cervical exfoliated cell sample in Example 4, wherein the detection signal type is 2R2G2A, and the detection signal type is 2R2G2A, and the detection result is: the TERC gene is not amplified, and the MYC gene is not expanded. increase.
  • Example 4 is a FISH detection result of the cervical tissue sample in Example 4, wherein the detection signal type is 2R2G2A, and the detection result is that the TERC gene is not amplified, and the MYC gene is not amplified.
  • Fig. 5 is a graph showing the results of FISH detection of cervical tissue samples in Example 4, wherein the detection signal type is 3R3G2A, and the detection results are: TERC gene amplification, MYC gene amplification.
  • a clone comprising the TERC of the gene of interest and the sequences at both ends was selected, as shown in Fig. 1A.
  • the GSP TERC comprises two sets of probes, the first set of probes comprising a first probe, a second probe and a third probe, as shown in the following table, purchased from the Invitrogen RP11 BAC and CTD BAC clone libraries.
  • the second set of probes included a fourth probe, a fifth probe, and a sixth probe, as specifically listed below, which were purchased from the Invitrogen RP11 BAC and CTD BAC clone libraries.
  • Second probe RP11-816J6 (chr3: 169447253..169494853) 48Kb
  • Third probe RP11-778I3 (chr3:169492904..169717073)224Kb
  • the GSP MYC includes two sets of probes:
  • the first set of probes included the first probe, as shown in the table below, which was purchased from the Invitrogen RP11 BAC and CTD BAC clone libraries.
  • the second set of probes included a second probe and a third probe, as detailed below, which were purchased from the Invitrogen RP11 BAC and CTD BAC clone libraries.
  • 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:
  • the red signal shows GSP TERC
  • the green signal shows GSP MYC
  • the cyan signal CSP 7 Chrosome 7 probe, purchased from D7Z1SpectrumAqua Probe, article number: 06J54-007. It can be seen that the TERC/MYC/7 chromosome probe signal is bright, and sensitivity and specificity can be observed on the metaphase chromosome in human peripheral blood cultured cell sheets. 100% sex; using a paraffin sample for hybridization detection, three target copy numbers can be clearly recorded. The results of other corresponding probe verification experiments are the same as those of the TERC group one probe + MYC group one probe, and the figures are omitted.
  • the TERC gene and MYC gene detection kit includes two components of a TERC/MYC hybridization solution and a DAPI counterstain, wherein the TERC and MYC hybridization solution comprises the set of GSP MYC gene probes described in Example 1 and a set of GSP TERCs. A combination of gene probes.
  • the TERC and MYC genes have two sets of detection probes respectively, and the two groups of the two genes can be combined at will.
  • the TERC gene and the MYC gene detection kit are four, respectively: TERC (group one) + Combination of MYC (Group 1), combination of TERC (Group 2) + MYC (Group 2), combination of TERC (Group 1) + MYC (Group 2), TERC (Group 2) + MYC (Group 1), CSP7 (Chromosome 7 probe, available from D7Z1 Spectrum Aqua Probe, Cat. No. 06J54-007), buffer component for hybridization environment (promoting hybridization), COT Human DNA with closed repeats, 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.
  • Example 3 Detection method of TERC gene and MYC gene detection kit
  • the relevant fluorescence and DAPI need to be observed with a suitable filter block.
  • Each negative control panel randomly counts the complete 100 cells, counts the number and percentage of abnormal signal cells in each sample, the average and standard deviation of the statistical percentage, and the negative threshold is set to the mean + 3 standard deviation .
  • Example 4 Clinical evaluation of TERC gene and MYC gene detection kit
  • Example 1 Using the TERC gene and MYC gene detection probe combination described in Example 1, the four detection kits described in Example 2 (TERC (Group 1) + MYC (Group 1) combination, TERC (Group 2) + MYC (Group) 2) combination, TERC (group 1) + MYC (group 2) combination, TERC (group 2) + MYC (group 1) combination) for 20 clinical samples (sample type is formalin fixed paraffin embedded) Tissue samples and cervical exfoliated cell samples were diagnosed by pathological examination, as shown in the table below, and tested separately. 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 and Figure 5 show the combined kit detection results of TERC (Group 1) + MYC (Group 1).
  • the detection signal type is 2R2G2A, and TERC/MYC is not amplified;
  • the detection signal type is 3R3G2A, and the TERC/MYC gene is amplified.
  • the detection results of the combinations of the other three different gene probes are the same, and the figures are omitted.
  • the detection of the TERC gene and the MYC gene by using one probe can also achieve the corresponding detection, but the use of the combined probe can be better than the combination of the probes, especially in the present embodiment.
  • the signal of TERC (group 1) + MYC (group 1) and TERC (group 2) + MYC (group 2) combination detection is the best. 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 BAC clones of the detection probes of Groups 1 and 2 for the TERC gene of the present invention have lengths of 564 Kb and 497 Kb, respectively, and are nucleic acid mixtures comprising the TERC gene and its both ends.
  • the total lengths of the BAC clones of the detection probes of Groups 1 and 2 for the MYC gene of the present invention are: 213 Kb and 291 Kb, respectively, which are nucleic acid mixtures comprising the MYC 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

La présente invention concerne une sonde de détection du gène TERC et du gène MYC, et son procédé de préparation. Le procédé comprend les étapes suivantes : sélection d'au moins l'un de CTD-3214J12, RP11-816J6 et RP11-778I3 comme clone BAC pour un gène TERC, ou sélection d'au moins l'un de RP11-3K16, CTD-2582E13 et RP11-886J24 comme clone BAC ; et/ou sélection de CTD-2511O8 comme clone BAC pour un gène MYC, ou sélection d'au moins l'un de RP11-440N18 et CTD-2205H22 comme clone BAC ; obtention d'un ADN plasmide ; exécution de l'étiquetage. La présente invention décrit également une trousse de réactifs comprenant la sonde de détection du gène TERC et du gène MYC.
PCT/CN2016/105711 2015-12-30 2016-11-14 Sonde de détection du gène terc et/ou du gène myc, son procédé de préparation, et trousse de réactifs Ceased WO2017114005A1 (fr)

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CN112877404A (zh) * 2020-08-12 2021-06-01 广州赛诚生物科技有限公司 一种检测体内rna与dna及蛋白互作的试剂盒及其使用方法

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CN120006001A (zh) * 2025-04-18 2025-05-16 北京大学人民医院 一种用于检测myc/mycn/mycl扩增的三色探针试剂及其应用

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