Probe for detecting deletion of glioma chromosome site 1P32, preparation method and use method thereof

Abstract

The present invention provides a probe for detecting deletions at the 1p32 chromosome locus in gliomas, as well as methods for preparing and using the probe. The CDKN2C and FAF1 genes represent 100% of the corresponding overall chromosome loss in 1p32 segment loss. Therefore, for detection at the 1p32 location, complementary gene segments must be designed to cover the transcriptional start ranges of both. By analyzing the gene segments at the 1p32 location, the transcriptional start regions of CDKN2C and FAF1, plus 20 bp downstream, were selected to design and synthesize corresponding fluorescent probes for detection and analysis. Furthermore, the present invention utilizes the centromere at the 1p chromosome location as a reference for chromosome copy number variation. Combined with the experimental techniques and methods provided by the present invention, the probe can rapidly and accurately detect chromosome copy number variation at the 1p32 location and determine whether the chromosome copy number at the 1p32 location has been amplified or lost.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a probe for detecting deletion of glioma chromosome site 1P32, and a preparation method and a use method thereof. Background Art

[0002] This section merely provides background information related to the present disclosure and is not necessarily prior art.

[0003] The short arm of chromosome 1 contains a high density of genes related to cell growth, proliferation, and differentiation. Loss of the short arm of chromosome 1 (1p) in gliomas is associated with a poor prognosis. Simultaneous loss of both the short arm of chromosome 1 and the long arm of chromosome 19 suggests a good prognosis.

[0004] The main detection methods currently on the market include fluorescence in situ hybridization (FISH) and next-generation sequencing (NGS). FISH is a commonly used non-radioactive in situ hybridization technique. It uses reporter molecules such as biotin and digoxin to label nucleic acid probes, and then detects hybrids of target DNA and nucleic acid probes through homologous complementarity between the probes and target DNA on chromosomes. The reporter molecules that form the hybrids, labeled with specific avidin and fluorescein, produce an immunochemical reaction. The fluorescence detection system allows for qualitative, quantitative, or relative positioning analysis of DNA under a microscope. Compared to next-generation sequencing, FISH offers advantages such as low cost, rapid detection, and accurate positioning.

[0005] The existing 1p chromosome deletion test on the market uses an in situ hybridization fluorescent probe to detect the local site 1p36 on 1p, but it needs to be combined with the position of 19q to determine the deletion status of 1p / 19q. Summary of the Invention

[0006] In view of the above problems, the first aspect of the present invention provides a method for preparing a probe for detecting deletion of the glioma chromosome site 1P32, comprising:

[0007] Nick translation was used to label the transcription start sites of CDKN2C and FAF1 between two exon regions plus 20 bp downstream. An equal volume of 0.1 mmol / L dNTP, 0.3 mmol / L dATP, 0.3 mmol / L dCTP, and 0.3 mmol / L dGTP was prepared. A 1-fold volume of 0.1 mmol / L dTTP and 2-fold volume of triple-distilled water were mixed. Nick translation experiments were performed using the following reaction system: 6.5 μl of 0.1 mmol / L dTTP, 10 μl of 0.1 mmol / L dNTP, 5 μl of 10× Nick Translation buffer, 0.5 μl of 1 mmol / L DIG-11-dUTP / Biotin-16-dUTP, 10 μl of Nick Translation Enzyme, X μl of DNA (1 μg), and 18-X μl of H2O, in a total of 50 μl.

[0008] The size of the probe was determined by gel electrophoresis. 5 μl of the sample was heated at 70°C for 5 minutes and then subjected to 2% agarose gel electrophoresis to observe the size of the probe. The probe was a single sequence probe with a size of 200-600 bp, indicating that the probe was of appropriate size. If the probe was too large, the reaction was extended at 15°C for 10-30 minutes, and the gel electrophoresis was repeated until the probe was 200-600 bp in size. 1 μl of 0.5 M EDTA was added to the reaction system and / or heated at 70°C for 5 minutes to inactivate the enzyme. The probe was stored at -20°C for later use.

[0009] The CDKN2C and FAF1 genes are 100% lost in the 1p32 segment loss, so detection at the 1p32 location requires the design of complementary gene segments that cover the transcriptional start range of both. By analyzing the gene segments at the 1p32 location, we selected the region between the two exons of CDKN2C and FAF1 transcriptional start plus 20bp downstream, and designed and synthesized corresponding fluorescent probes for detection and analysis. Furthermore, the present invention utilizes the centromere at the 1p chromosome location as a reference for chromosome copy number variation. Combined with the experimental techniques provided by the present invention, the probe can quickly and accurately detect chromosome copy number variation at the 1p32 location and determine whether the chromosome copy number at the 1p32 location has been amplified or lost.

[0010] The present invention focuses on the characteristics of partial chromosomal deletion at the 1p chromosome position of glioma. Through screening, it is discovered that the 1p32 position has special value independent of the original traditional 1p36 position determination. A corresponding probe is provided as well as corresponding preparation and use methods and statistical determination methods.

[0011] The TCGA database shows that the loss of 1p32 and 1p36 do not completely overlap, and there are corresponding differences between them. At the same time, the loss of 1p32 shows a prognostic impact independent of 1p36. Therefore, the detection of 1p32 chromosome deletion has important diagnostic and detection value in glioma. The existing market uses a 1p36 site detection kit for the judgment of 1p chromosome. The present invention uses FISH probe technology to design and develop corresponding probes based on gene sites, which supplements the detection method of glioma chromosome copy number variation and has a strong application value for promotion.

[0012] In some embodiments of the present invention, the reaction system is used to label 1 μg of DNA. When a larger amount of DNA needs to be labeled, the amount of reagents and the reaction system are correspondingly expanded by equal folds.

[0013] In some embodiments of the present invention, the components are mixed and then centrifuged briefly. For plasmids ≤ 10 kb, the reaction is carried out at 15° C. for 3.5 hours, and for BAC / PAC, the reaction is carried out at 15° C. for 9 hours.

[0014] The second aspect of the present invention provides a probe for detecting deletion of glioma chromosome site 1P32, which is prepared by the method for preparing a probe for detecting deletion of glioma chromosome site 1P32 described in the above technical solution.

[0015] The beneficial effects of the probe for detecting deletion of glioma chromosome site 1P32 in the embodiment of the present invention and the preparation method of the probe for detecting deletion of glioma chromosome site 1P32 in the above embodiment are the same, which will not be repeated here.

[0016] The third aspect of the present invention provides a method for using a probe for detecting deletions at the 1P32 chromosome site in gliomas, wherein the method comprises:

[0017] Prepare experimental solutions;

[0018] Pre-process tissue samples;

[0019] The tissue sample is probed using a probe.

[0020] The beneficial effects of the probe for detecting deletion of glioma chromosome site 1P32 in the embodiment of the present invention and the preparation method of the probe for detecting deletion of glioma chromosome site 1P32 in the above embodiment are the same, which will not be repeated here.

[0021] In some embodiments of the present invention, preparing the experimental solution comprises:

[0022] Prepare NaOH solution: a) Weigh 4 g of analytically pure NaOH solid using a balance and place it in an Erlenmeyer flask; b) Use a 200 ml graduated cylinder to accurately measure 100 ml of distilled water and slowly pour it into the Erlenmeyer flask containing the NaOH solid; c) Shake the Erlenmeyer flask to fully dissolve the NaOH solid; d) Then, slowly pour the solution into a 250 ml wide-mouth bottle, seal, and store until ready for use.

[0023] Prepare 20X SSC solution: add 175.5 g sodium chloride and 88.2 g trisodium citrate to 1000 ml of double-distilled water and stir with a magnetic stir bar until completely dissolved. Filter through filter paper and adjust the pH to 5.3. Seal with parafilm and store at 4°C until needed. The shelf life is 6 months.

[0024] Prepare 2X SSC solution: dilute 20X SSC and double-distilled water in a ratio of 1:9, adjust the pH to 7.0, seal with parafilm, and store at 4°C until use. The shelf life is 6 months.

[0025] Prepare 2x SSC / 0.1% NP-40:

[0026] 50 ml 20×SSC (pH 5.3)

[0027] 0.5ml NP-40

[0028] 449.5ml double-distilled water

[0029] Prepare 500 ml, adjust the pH to 7.0, seal with parafilm, and store at room temperature for future use. The shelf life is 6 months.

[0030] Prepare a series of alcohols: Take 28 ml and 34 ml of anhydrous ethanol, mix them with 12 ml and 6 ml of double-distilled water, respectively, to make 70% and 85% alcohols, and combine them with anhydrous ethanol to make 70%, 85%, and 100% series of alcohols. Seal the bottles with parafilm and store at room temperature until used. Prepare another batch of the above series of alcohols, seal them with parafilm, and store at -20°C until used.

[0031] Prepare PBS buffer:

[0032] PBS 1 L formula (pH 7.4): Potassium dihydrogen phosphate (KH2PO4): 0.27g

[0033] Sodium hydrogen phosphate (Na2HPO4): 1.42g

[0034] Sodium chloride (NaCl): 8g

[0035] Potassium chloride (KCl) 0.2g

[0036] Add about 800 mL of deionized water and stir thoroughly to dissolve. Then add concentrated hydrochloric acid to adjust the pH to 7.4. Finally, make up to 1 L. Sterilize at high temperature and high pressure and store at room temperature.

[0037] Prepare 0.075 M KCl hypotonic solution: dissolve 5.59 g potassium (KCl) in 1000 ml deionized water and store at 4°C or dissolve 2.794 g potassium (KCl) in 500 ml deionized water and store at 4°C.

[0038] Prepare the fixative solution: Prepare the cell fixative solution at a ratio of methanol (AR) to glacial acetic acid (AR) = 3:1. Prepare the cell fixative solution and use it immediately.

[0039] Prepare 1M HCl: Take 0.9 ml of concentrated HCl and add deionized water to 100 ml, mix well, and store at 4°C until use. Keep for 1 month.

[0040] Prepare a 0.08 mg / ml pepsin working solution: a) Dissolve 40 mg of pepsin in a 1.5 ml EP tube, add 1 ml of deionized water, and aliquot into ten 200 μl centrifuge tubes. b) To reuse, add 50 ml of deionized water to a Coplin bottle, add 500 μl of 1 M HCl, and then add one aliquot of pepsin solution (100 μl).

[0041] Prepare 200 μg / ml PK working solution: a PK storage solution (20 mg / ml): Weigh 0.1 g of proteinase K dry powder and dissolve it in 2X SSC (pH = 7.0) solution. Gently shake until the PK is completely dissolved. Do not vortex to mix. Store at -20°C. b Proteinase K working solution (200 μg / ml): Take 0.4 ml of PK storage solution and dissolve it in 40 ml of 2X SSC (pH 7.0) solution.

[0042] In some embodiments of the present invention, pre-processing the tissue sample includes:

[0043] Baking: Bake the tissue sections at 65°C overnight to age the slides;

[0044] Dewaxing: Immerse the tissue sections in xylene for dewaxing twice at room temperature, 10 min each time;

[0045] Washing: Immerse the tissue sections in 100% ethanol for 5 minutes;

[0046] Gradient rehydration: the tissue sections were sequentially rehydrated in 100%, 85%, and 70% ethanol at room temperature for 2 min each;

[0047] Protein removal: the tissue sections were placed in deionized water and incubated at 90°C for 30 min;

[0048] Prepare pepsin working solution: add (3.2 mg x 3 = 9.6 mg) pepsin to 40 ml 0.01 M HCl to make pepsin working solution, and incubate at 37°C for 25-30 min.

[0049] Rinse: Rinse twice in 2XSSC solution, 5 minutes each time;

[0050] Air drying: Place the slides in 70% and 95% ethanol for 2 minutes each and air dry the slides.

[0051] In some embodiments of the present invention, detecting the tissue sample using a probe comprises:

[0052] Prepare a probe mixture at a ratio of 2:8, drop 10 μl of the probe mixture onto the hybridization area of ​​the slide, cover with a small slide and mounting glue, denature at 82°C for 5 minutes on a hybridizer, and hybridize overnight.

[0053] Remove the sealing glue and small glass slides, and place in a 2XSSC solution at 46°C (46-48°C) in a water bath for 5 min, double;

[0054] Place the slide in 46°C 0.1% NP-40 / 2XSSC, shake for 1-3 seconds, and remove after 5 minutes;

[0055] Dehydrate in 70% and 95% ethanol for 2 min each, spin dry, and dry naturally;

[0056] After drying, the slides were allowed to air dry, 15 μl of DAPI was added to the hybridization area, and a large coverslip was placed on the slides, and the slides were observed under a fluorescence microscope after 20 min;

[0057] Observation results: The centromere site is a red signal, the detection signal of 1p32 is a green signal, and the red signal in the nucleus indicates the total number of 1p chromosomes. If the number of green signals in the cell nucleus is equal to the red signal, no loss has occurred; if the number of green signals is less than the red signal, the tissue sample is 1p32 LOH (loss of chromosome signal at 1p32 position); if the number of green signals is greater than the red signal, the tissue sample is 1p32 AMP (amplification of chromosome signal at 1p32 position).

[0058] In some embodiments of the present invention, in the pre-processing of the tissue sample,

[0059] Dewaxing: Place the tissue sections in a bottle of xylene for dewaxing for 20-30 minutes;

[0060] Protein removal: the tissue slices were treated with 30% w / v sodium sulfite at 50° C. for 20-30 minutes;

[0061] Prepare pepsin working solution: take (3.2 mg x 3 = 9.6 mg) pepsin and add 200 ug / ml PK for 10 min.

[0062] In some embodiments of the present invention, after rinsing twice in 2XSSC solution for 5 minutes each time, the method of using the probe for detecting deletion of glioma chromosome locus 1P32 further comprises immersing the rinsed slide in PBS buffer containing 2.5% formaldehyde for 10 minutes. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Various other advantages and benefits will become apparent to those skilled in the art upon reading the detailed description of the preferred embodiment below. The accompanying drawings are for illustration purposes only and are not to be considered as limiting the present invention. The same reference symbols are used throughout the drawings to represent the same components. In the drawings:

[0064] Figure 1 Schematic diagram of the probe design for the 1p32 locus according to an embodiment of the present invention;

[0065] Figure 2 It is one of the most common deletion sites in low-grade gliomas;

[0066] Figure 3 for Figure 2 The enlarged view of the part shown;

[0067] Figure 4 It is the intersection and difference map of 19q13, 1p32 and 1p36 loci;

[0068] Figure 5 Schematic diagram of multivariate statistical analysis of overall survival;

[0069] Figure 6 This is the result of the 1p32 locus probe detection. DETAILED DESCRIPTION

[0070] Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments described herein. Rather, these embodiments are provided to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art.

[0071] It should be understood that the terms used herein are for the purpose of describing specific example embodiments only and are not intended to be limiting. Unless the context clearly indicates otherwise, as used herein, the singular forms "a," "an," and "the" may also be intended to include the plural forms. The terms "comprise," "include," "contain," and "have" are inclusive and, therefore, specify the presence of the stated features, steps, operations, elements, and / or parts, but do not preclude the presence or addition of one or more other features, steps, operations, elements, parts, and / or combinations thereof.

[0072] Although the terms first, second, third, etc. can be used in the text to describe multiple elements, components, regions, layers and / or sections, these elements, components, regions, layers and / or sections should not be limited by these terms. These terms can only be used to distinguish an element, component, region, layer or section from another region, layer or section. Unless the context clearly indicates otherwise, terms such as "first", "second" and other numerical terms do not imply order or sequence when used in the text. Therefore, the first element, component, region, layer or section discussed below can be referred to as the second element, component, region, layer or section without departing from the teaching of the example embodiments.

[0073] For ease of description, spatially relative terms may be used herein to describe the relationship of one element or feature relative to another element or feature as shown in the figures, such as "inside," "outside," "inside," "outside," "below," "beneath," "above," and the like. Such spatially relative terms are intended to include different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures is flipped, an element described as "below" or "below" another element or feature would then be oriented as "above" or "above" another element or feature. Thus, the example term "below" can include both above and below orientations. The device may be otherwise oriented (rotated 90 degrees or in other orientations) and the spatially relative descriptors used herein are interpreted accordingly.

[0074] The disclosure below provides many different embodiments or examples for realizing different structures of the present invention. In order to simplify the disclosure of the present invention, the components and settings of specific examples are described below. Of course, they are merely examples and are not intended to limit the present invention. In addition, the present invention may repeat reference numbers and / or reference letters in different examples. Such repetition is for the purpose of simplicity and clarity and does not in itself indicate the relationship between the various embodiments and / or settings discussed. In addition, the present invention provides examples of various specific processes and materials, but those skilled in the art will recognize the application of other processes and / or the use of other materials.

[0075] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0076] like Figures 1 to 6 As shown, the first aspect of the present invention provides a method for preparing a probe for detecting deletion of glioma chromosome site 1P32, comprising:

[0077] Nick translation was used to label the transcription start sites of CDKN2C and FAF1 between two exon regions plus 20 bp downstream. An equal volume of 0.1 mmol / L dNTP, 0.3 mmol / L dATP, 0.3 mmol / L dCTP, and 0.3 mmol / L dGTP was prepared. A 1-fold volume of 0.1 mmol / L dTTP and 2-fold volume of triple-distilled water were mixed. Nick translation experiments were performed using the following reaction system: 6.5 μl of 0.1 mmol / L dTTP, 10 μl of 0.1 mmol / L dNTP, 5 μl of 10× Nick Translation buffer, 0.5 μl of 1 mmol / L DIG-11-dUTP / Biotin-16-dUTP, 10 μl of Nick Translation Enzyme, X μl of DNA (1 μg), and 18-X μl of H2O, in a total of 50 μl.

[0078] The size of the probe was determined by gel electrophoresis. 5 μl of the sample was heated at 70°C for 5 minutes and then subjected to 2% agarose gel electrophoresis to observe the size of the probe. The probe was a single sequence probe with a size of 200-600 bp, indicating that the probe was of appropriate size. If the probe was too large, the reaction was extended at 15°C for 10-30 minutes, and the gel electrophoresis was repeated until the probe was 200-600 bp in size. 1 μl of 0.5 M EDTA was added to the reaction system and / or heated at 70°C for 5 minutes to inactivate the enzyme. The probe was stored at -20°C for later use.

[0079] like Figure 1 As shown, the CDKN2C and FAF1 genes are 100% lost in the 1p32 segment loss, so detection at the 1p32 location requires the design of complementary gene segments that cover the transcription start range of both. By analyzing the gene segments at the 1p32 location, we selected the region between the two exons of CDKN2C and FAF1 transcription start plus 20bp downstream, and designed and synthesized corresponding fluorescent probes for detection and analysis. Furthermore, the present invention utilizes the centromere at the 1p chromosome location as a reference for chromosome copy number variation. Combined with the experimental techniques provided by the present invention, the probe can quickly and accurately detect chromosome copy number variation at the 1p32 location and determine whether the chromosome at the 1p32 location has been amplified or lost.

[0080] The present invention focuses on the characteristics of partial chromosomal deletion at the 1p chromosome position of glioma. Through screening, it is discovered that the 1p32 position has special value independent of the original traditional 1p36 position determination. A corresponding probe is provided as well as corresponding preparation and use methods and statistical determination methods.

[0081] like Figure 4 and Figure 5 As shown in the TCGA database, the loss of 1p32 and 1p36 do not completely overlap, and there are corresponding differences between them. At the same time, the loss of 1p32 shows a prognostic impact independent of 1p36. Therefore, the detection of 1p32 chromosome deletion has important diagnostic and detection value in glioma. The existing market uses a 1p36 site detection kit for the judgment of 1p chromosome. The present invention uses FISH probe technology to design and develop corresponding probes according to the gene site, which supplements the detection method of glioma chromosome copy number variation and has a strong application value for promotion.

[0082] In some embodiments of the present invention, the reaction system is used to label 1 μg of DNA. When a larger amount of DNA needs to be labeled, the amount of reagents and the reaction system are correspondingly expanded by equal folds.

[0083] In some embodiments of the present invention, the components are mixed and then centrifuged briefly. For plasmids ≤ 10 kb, the reaction is carried out at 15° C. for 3.5 hours, and for BAC / PAC, the reaction is carried out at 15° C. for 9 hours.

[0084] The second aspect of the present invention provides a probe for detecting deletion of glioma chromosome site 1P32, which is prepared by the method for preparing a probe for detecting deletion of glioma chromosome site 1P32 described in the above embodiment.

[0085] like Figure 6As shown, the third aspect of the present invention provides a method for using a probe for detecting deletion of the glioma chromosome site 1P32, using the probe for detecting deletion of the glioma chromosome site 1P32 described in the above embodiment, comprising:

[0086] Prepare experimental solutions;

[0087] Pre-process tissue samples;

[0088] The tissue sample is probed using a probe.

[0089] In some embodiments of the present invention, preparing the experimental solution comprises:

[0090] Prepare NaOH solution: a) Weigh 4 g of analytically pure NaOH solid using a balance and place it in an Erlenmeyer flask; b) Use a 200 ml graduated cylinder to accurately measure 100 ml of distilled water and slowly pour it into the Erlenmeyer flask containing the NaOH solid; c) Shake the Erlenmeyer flask to fully dissolve the NaOH solid; d) Then, slowly pour the solution into a 250 ml wide-mouth bottle, seal, and store until ready for use.

[0091] Prepare 20X SSC solution: add 175.5 g sodium chloride and 88.2 g trisodium citrate to 1000 ml of double-distilled water and stir with a magnetic stir bar until completely dissolved. Filter through filter paper and adjust the pH to 5.3. Seal with parafilm and store at 4°C until needed. The shelf life is 6 months.

[0092] Prepare 2X SSC solution: dilute 20X SSC and double-distilled water in a ratio of 1:9, adjust the pH to 7.0, seal with parafilm, and store at 4°C until use. The shelf life is 6 months.

[0093] Prepare 2x SSC / 0.1% NP-40:

[0094] 50 ml 20×SSC (pH 5.3)

[0095] 0.5ml NP-40

[0096] 449.5ml double-distilled water

[0097] Prepare 500 ml, adjust the pH to 7.0, seal with parafilm, and store at room temperature for future use. The shelf life is 6 months.

[0098] Prepare a series of alcohols: Take 28 ml and 34 ml of anhydrous ethanol, mix them with 12 ml and 6 ml of double-distilled water, respectively, to make 70% and 85% alcohols, and combine them with anhydrous ethanol to make 70%, 85%, and 100% series of alcohols. Seal the bottles with parafilm and store at room temperature until used. Prepare another batch of the above series of alcohols, seal them with parafilm, and store at -20°C until used.

[0099] Prepare PBS buffer:

[0100] PBS 1 L formula (pH 7.4): Potassium dihydrogen phosphate (KH2PO4): 0.27g

[0101] Sodium hydrogen phosphate (Na2HPO4): 1.42g

[0102] Sodium chloride (NaCl): 8g

[0103] Potassium chloride (KCl) 0.2g

[0104] Add about 800 mL of deionized water and stir thoroughly to dissolve. Then add concentrated hydrochloric acid to adjust the pH to 7.4. Finally, make up to 1 L. Sterilize at high temperature and high pressure and store at room temperature.

[0105] Prepare 0.075 M KCl hypotonic solution: dissolve 5.59 g potassium (KCl) in 1000 ml deionized water and store at 4°C or dissolve 2.794 g potassium (KCl) in 500 ml deionized water and store at 4°C.

[0106] Prepare the fixative solution: Prepare the cell fixative solution at a ratio of methanol (AR) to glacial acetic acid (AR) = 3:1. Prepare the cell fixative solution and use it immediately.

[0107] Prepare 1M HCl: Take 0.9 ml of concentrated HCl and add deionized water to 100 ml, mix well, and store at 4°C until use. Keep for 1 month.

[0108] Prepare a 0.08 mg / ml pepsin working solution: a) Dissolve 40 mg of pepsin in a 1.5 ml EP tube, add 1 ml of deionized water, and aliquot into ten 200 μl centrifuge tubes. b) To reuse, add 50 ml of deionized water to a Coplin bottle, add 500 μl of 1 M HCl, and then add one aliquot of pepsin solution (100 μl).

[0109] Prepare 200 μg / ml PK working solution: a PK storage solution (20 mg / ml): Weigh 0.1 g of proteinase K dry powder and dissolve it in 2X SSC (pH = 7.0) solution. Gently shake until the PK is completely dissolved. Do not vortex to mix. Store at -20°C. b Proteinase K working solution (200 μg / ml): Take 0.4 ml of PK storage solution and dissolve it in 40 ml of 2X SSC (pH 7.0) solution.

[0110] In some embodiments of the present invention, pre-processing the tissue sample includes:

[0111] Baking: Bake the tissue sections at 65°C overnight to age the slides;

[0112] Dewaxing: Immerse the tissue sections in xylene for dewaxing twice at room temperature, 10 min each time;

[0113] Washing: Immerse the tissue sections in 100% ethanol for 5 minutes;

[0114] Gradient rehydration: rehydrate the tissue sections in 100%, 85%, and 70% ethanol at room temperature for 2 minutes each;

[0115] Deproteinization: Incubate tissue sections in deionized water at 90°C for 30 min.

[0116] Prepare pepsin working solution: add (3.2 mg x 3 = 9.6 mg) pepsin to 40 ml 0.01 M HCl to make pepsin working solution, and incubate at 37°C for 25-30 min.

[0117] Rinse: Rinse twice in 2XSSC solution, 5 minutes each time;

[0118] Air-drying: Place the slides in 70% and 95% ethanol for 2 minutes each and air-dry. This step involves washing the xylene with 100% ethanol and then using deionized water to remove surface proteins from the nucleic acids, increase tissue permeability, facilitate probe hybridization, and improve detection accuracy and speed.

[0119] In some embodiments of the present invention, detecting the tissue sample using a probe comprises:

[0120] Prepare a probe mixture at a ratio of 2:8, drop 10 μl of the probe mixture onto the hybridization area of ​​the slide, cover with a small slide and mounting glue, denature at 82°C for 5 minutes on a hybridizer, and hybridize overnight.

[0121] Remove the sealing glue and small glass slides, and place in a 2XSSC solution at 46°C (46-48°C) in a water bath for 5 min, double;

[0122] Place the slide in 46°C 0.1% NP-40 / 2XSSC, shake for 1-3 seconds, and remove after 5 minutes;

[0123] Dehydrate in 70% and 95% ethanol for 2 min each, spin dry, and dry naturally;

[0124] After drying, the slides were allowed to air dry, 15 μl of DAPI was added to the hybridization area, and a large coverslip was placed on the slides, and the slides were observed under a fluorescence microscope after 20 min;

[0125] Observation results: The central centromere site is a red signal, and the detection signal of 1p32 is a green signal. The red signal in the nucleus indicates the total number of 1p chromosomes. If the number of green signals in the cell nucleus is equal to the red signal, no loss has occurred; if the number of green signals is less than the red signal, the sample is 1p32 LOH (loss of chromosome signal at 1p32 position); if the number of green signals is greater than the red signal, the sample is 1p32 AMP (amplification of chromosome signal at 1p32 position).

[0126] In some embodiments of the present invention, in the pre-processing of the tissue sample,

[0127] Dewaxing: Place the tissue sections in a bottle of xylene for 20-30 minutes;

[0128] Deproteinization: Treat tissue sections with 30% w / v sodium sulfite at 50°C for 20-30 minutes.

[0129] Prepare pepsin working solution: take (3.2 mg x 3 = 9.6 mg) pepsin and add 200 ug / ml PK for 10 min.

[0130] In some embodiments of the present invention, after rinsing twice in 2XSSC solution for 5 minutes each time, the method of using the probe for detecting deletion of glioma chromosome locus 1P32 further comprises immersing the rinsed slide in PBS buffer containing 2.5% formaldehyde for 10 minutes.

[0131] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. Use of a reagent for detecting deletion of chromosome locus 1P32 in the preparation of a product for detecting glioma prognosis, characterized in that: The reagent for detecting the deletion of the chromosome site 1P32 includes a probe for detecting the deletion of the chromosome site 1P32, and the probe preparation steps include: Nick translation was used to label the transcription start sites of CDKN2C and FAF1 between two exons plus 20 bp downstream. An equal volume of 0.1 mmol / L dNTP, 0.3 mmol / L dATP, 0.3 mmol / L dCTP, and 0.3 mmol / L dGTP was mixed. A 1-fold volume of 0.1 mmol / L dTTP and 2-fold volume of triple-distilled water were mixed. Nick translation experiments were performed using the following reaction system: 6.5 μl of 0.1 mmol / L dTTP, 10 μl of 0.1 mmol / L dNTP, 5 μl of 10× Nick Translation buffer, 0.5 μl of 1 mmol / L DIG-11-dUTP / Biotin-16-dUTP, 10 μl of Nick Translation Enzyme, X μl of DNA, and 18-X μl of H2O, in a total of 50 μl. The size of the probe was determined by gel electrophoresis. 5 μl of the EP tube was placed on ice, heated at 70°C for 5 minutes, and then subjected to 2% agarose gel electrophoresis to observe the size of the probe. The probe was a single sequence probe with a size of 200-600 bp, indicating that the probe was of appropriate size. If the probe was too large, the reaction was extended at 15°C for 10-30 minutes, and the gel electrophoresis was repeated until the probe was 200-600 bp in size. 1 μl of 0.5 M EDTA was added to the reaction system and / or heated at 70°C for 5 minutes to inactivate the enzyme. The probe was stored at -20°C for later use.

2. Use of the reagent for detecting the deletion of chromosome locus 1P32 according to claim 1 in the preparation of a product for detecting the prognosis of glioma, characterized in that: The reaction system is used to label 1 μg of DNA. When a larger amount of DNA needs to be labeled, the amount of reagents and the reaction system are correspondingly expanded by the same multiple.

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