A simple method for chromosome pressing combined with in situ hybridization

Abstract

The present invention belongs to the field of molecular cell genetics and chromosome engineering technology, and specifically relates to a simple operation method for chromosome pressing combined with in situ hybridization. The present invention further uses 4% paraformaldehyde treatment for double fixation on the basis of methanol glacial acetic acid fixation of chromosome pressing, so that the chromosome structure morphology can withstand high-temperature deformation above 90 degrees, and can provide a clearer chromosome morphological structure compared with traditional methods. At the same time, the present invention also develops a corresponding universal hybridization solution system and rinsing solution system, and there is no need to explore various complicated hybridization annealing processes and rinsing processes due to different probes. Compared with the traditional chromosome pressing combined with in situ hybridization technology, the present invention has higher chromosome structure morphology clarity, while maintaining the hybridization signal, the chromosome break position can still be clearly observed in the non-signal area, which helps to provide technical support for chromosome-related genetic analysis research.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular cell genetics and chromosome engineering, and in particular relates to a simple operation method for chromosome pressing combined with in situ hybridization. Background Art

[0002] Chromosome flattening technology and related derivative technologies are the basis of chromosome cytogenetics analysis and research. Chromosome flattening technology is a microscope sectioning technology that prepares chromosomes by fixing, staining and flattening cells. It is widely used in cytogenetics, gene localization and chromosome abnormality analysis. At present, the commonly used chromosome karyotype banding technology originated from the plant chromosome banding flattening technology. Its basic operation steps include: collecting cell tissue samples in the division period, fixing the cell division state with a fixative, lysing the cells to release chromosomes, and marking DNA with a dye; then, unfolding the chromosome flattening on a glass slide, and observing its number, morphology and structural changes under a microscope. This technology is not only used to study the typing and identification of genetic abnormalities such as chromosome rearrangement and deletion, but also can be combined with in situ hybridization technology (such as FISH) to locate genes or repetitive sequences, and help analyze chromosome behavior in cell division. It is irreplaceable in chromosome behavior research (such as mitosis and meiosis).

[0003] In situ hybridization is a method used to detect the spatial distribution and location of specific DNA or RNA sequences in cells or tissues. It hybridizes labeled probes (usually nucleic acid probes) with target sequences in samples to accurately locate target nucleic acid molecules. The operation process of this technology usually includes: preparing samples containing target nucleic acids, hybridizing with labeled probes after fixation and permeabilization, and determining the spatial distribution of nucleic acids through microscopic imaging. This technology can be used to locate specific genes, analyze gene expression patterns, detect chromosomal abnormalities, and study the distribution and function of RNA, etc., providing important technical support for molecular and cell biology research.

[0004] Combining chromosome squeezing and in situ hybridization technology can provide higher resolution and more accurate chromosome sequence positioning. It is a classic and important core technology in chromosome research, especially for highly repetitive heterochromatin regions in the genome. Because it is very challenging and difficult to achieve in this region to locate these repetitive sequences and construct a genome sequence framework using genome sequencing technology combined with computational analysis. However, chromosome squeezing combined with in situ hybridization technology is still the most effective method for locating these highly repetitive sequences at the subchromosome level, and it is also an important core means for experimental verification and positioning of a large number of repetitive sequences in genome and transcriptome sequencing research. However, due to the influence of probe types and the need for specific experimental operations for each probe, the current in situ hybridization technology can only detect aneuploidy of chromosomes 13, 18, 21, X, and Y, and does not cover other chromosome number abnormalities. At the same time, due to the high difficulty of experimental operation, it can only be detected in the interphase of cells, resulting in the loss of a lot of relevant information about chromosome abnormalities. In addition, specific chromosome abnormalities, such as chromosome breakage and translocation, have inconsistent results due to the high difficulty of the experiment and the lack of uniform operation procedures. Therefore, developing a simple, efficient, scalable and easy-to-use chromosome pressing combined with in situ hybridization detection method has important research and application value.

[0005] The current chromosome pressing combined with in situ hybridization technology has a cumbersome operation process and requires high experimental experience and operation skills. Its difficulties are mainly reflected in the following aspects: (1) It is necessary to prepare a good chromosome fixed sample to ensure that the sample is fully unfolded in the middle and late stages of cell division. Any improper fixation and handling may cause chromosome structure distortion, thereby affecting the hybridization effect. In addition, the chromosome fixative and processing temperature need to be strictly controlled to avoid damaging the chromosome or reducing the hybridization efficiency. (2) Reasonable hybridization probe design and labeling are required: the length, sequence specificity and selection of fluorescent dyes of the probe need to be considered. If the probe design is inaccurate or the labeling effect is poor, it may lead to weak signal or non-specific binding. (3) The hybridization conditions need to be optimized: the hybridization temperature, time and salt concentration and other conditions need to be optimized to ensure the specific binding of the probe to the target sequence. Because different probes need to be adapted to different experimental systems, operators cannot obtain a unified operation process, and the success rate of the experiment is not guaranteed. For example, the traditional hybridization solution formula usually contains 10%-50% formamide, 5-20% dextran sulfate, 2-5×SSC, 0.1-0.5% (vol / vol) detergent (Tween 20, NP40, TritonX-100, etc.), 0.5%-5% BSA, blocked tRNA, blocked salmon sperm DNA, etc., and the applicable probes and hybridization temperatures are different according to the hybridization solution formula, and the denaturation temperature is mostly between 60-75 degrees. Therefore, the traditional hybridization solution is a highly variable formula system, and in order to adapt to some special processing samples, corresponding special reagents are often further added. Such variability brings difficulties to specific experimental operations. (4) Reasonable washing is required to remove non-specific binding: The washing step after hybridization is particularly important. Unbound probes must be removed to reduce background signals and improve signal clarity. Insufficient washing may lead to high background or incorrect signal positioning. For example, the traditional rinsing solution formula (taking the SSC buffer system as an example) usually contains 0.1-2×SSC, 0.1-0.5% (vol / vol) detergent (Tween 20, NP40, TritonX-100, etc.), 10%-50% formamide, etc. The temperature is generally room temperature to 55-60 degrees (lower than the hybridization temperature, which needs to be optimized according to different systems), and the rinsing process is very cumbersome and uncertain, which brings difficulties to the operator. It can be seen that the current chromosome pressing combined with in situ hybridization technology requires that each step must be carefully controlled and targetedly optimized during the experimental operation in order to obtain accurate and reliable experimental results. This has resulted in thousands of methods for thousands of people, which are difficult to unify, resulting in the inability to popularize and promote application, and therefore has extremely high requirements for the skills and experience of operators.

[0006] In summary, in view of the cumbersome operation and high threshold of experimental operation requirements of the traditional chromosome pressing combined with in situ hybridization technology, it is necessary to develop a universal and simple operation method to adapt to the implementation of different operators (especially experimental novices) and produce stable experimental results. Summary of the invention

[0007] In order to overcome the deficiencies of the above-mentioned prior art, the present invention aims at the current difficulties faced by chromosome compression combined with in situ hybridization technology, and proposes a general and simple operation method, which only requires simple laboratory equipment and reagents, and is very suitable for researchers who lack relevant operation experience. Specifically, the present invention improves the chromosome fixation method (using methanol glacial acetic acid + 4% paraformaldehyde double fixation method) so that the chromosome structure can resist high temperature deformation during the probe hybridization process, so that it can provide a clearer chromosome morphology structure than the traditional method, and can still clearly distinguish the chromosome breakpoints from the chromosome structure morphology while ensuring the hybridization signal. At the same time, due to the change in the fixation method of the chromosome morphology, the chromosome structure can withstand high temperatures above 90 degrees, thereby further reducing the difficulty of probe design (such as probe length, consideration of nonspecific binding, and subsequent processing of nonspecific binding signals). In addition, the present invention has also developed a corresponding hybridization solution formula (50% formamide, 10% dextran sulfate, 2×SSC, 0.5 μM of each probe) and a hybridization condition system, which only requires 91-95 degrees denaturation for 2-5 minutes, and then annealing to room temperature to complete the hybridization process, without the need to explore the corresponding annealing temperature for each probe, nor the need for overnight hybridization, and no need to purchase an expensive hybridizer. In addition, the present invention has also developed a matching simple rinse solution formula (0.1×SSC), which only requires room temperature to complete the rinsing process, avoiding the need for multiple formamide solution washing after hybridization in the traditional scheme, and the need to explore the rinsing temperature and other problems. In addition, the present invention only requires the use of a commercially synthesized DNA probe labeled with a single fluorescent molecule, which has high sensitivity. For the visualization of low copy number sequences, biotinylated long probes can also be used to improve the detection sensitivity through the cascade amplification effect of multiple biotin treatments, which provides technical support for the location study of non-repetitive sequence chromosomes, and also provides a reference for the establishment and application of a detection scheme combining chromosome karyotype analysis with in situ hybridization technology, which is conducive to promoting the development of chromosome cytogenetic analysis and related research fields.

[0008] In order to achieve the above object, the specific technical solution adopted by the present invention is as follows:

[0009] The present invention provides a simple operation method for chromosome pressing combined with in situ hybridization for non-disease diagnosis purposes, the method comprising the following steps:

[0010] S1. Treat the brain tissue with hypotonic buffer at room temperature for 3-10 minutes;

[0011] S2, fixing with a fixing solution at room temperature for 10-20 seconds, wherein the fixing solution comprises acetic acid, methanol and water;

[0012] S3, add 40%-50% acetic acid to the siliconized coverslip, transfer the brain tissue to 40%-50% acetic acid, and cover with a non-siliconized slide;

[0013] S4. Press down the coverslip firmly to spread out the brain tissue and stretch out the chromosomes.

[0014] S5. Treat the sample with liquid nitrogen, lift the cover glass, and place it in anhydrous ethanol for 10-30 minutes or overnight;

[0015] S6. Take out the slide and dry it at room temperature;

[0016] S7, wash the sample with 2×SSC, dehydrate in 60%-80% ethanol for 7-15 minutes, then treat in 90%-97% ethanol for 3-7 minutes, and dry;

[0017] S8. Treat the sample with 4% paraformaldehyde at room temperature for 20-50 min;

[0018] S9, rinse with PBS and dry;

[0019] S10, adding hybridization solution to the sample and covering the cover slip, wherein the hybridization solution includes 50% formamide, 10% dextran sulfate, 2×SSC, and 0.5 μM probe;

[0020] S11, denature at 90-95℃ for 2-5min; generally, the longer the probe, the higher the temperature and the longer the denaturation time. The higher the temperature and the longer the time, the smaller the nonspecific signal.

[0021] S12, then quickly cooled to a room temperature, and incubated in a humid dark light at room temperature for 0.5-2 hours or overnight; no specific annealing temperature is required for any probe, and all are annealed to room temperature;

[0022] S13, wash with 0.1×SSC 2-5 times, 10-20 minutes each time; no need to find a specific rinse temperature, room temperature can be used;

[0023] S14, stain with DAPI for 3-10 minutes;

[0024] S15, quickly wash with 2×SSC and allow the slide to dry;

[0025] S16. Cover the slides with Vectashield and analyze using a microscope.

[0026] The present invention improves the fixing method during chromosome pressing so that the chromosome structure and morphology can withstand high temperature deformation above 90 degrees, and can provide a clearer chromosome morphology structure compared with the traditional method. At the same time, the present invention also develops a corresponding universal hybridization solution system and rinsing solution system, which does not require the exploration of various complex hybridization annealing processes and rinsing processes due to different probes, thereby establishing a universal (any hybridization probe) and simple operation (especially friendly to novices) chromosome pressing combined with in situ hybridization detection method, which helps to provide technical support for chromosome-related genetic analysis research.

[0027] Preferably, the hypotonic buffer in S1 is 0.4%-0.6% sodium citrate dihydrate solution.

[0028] Preferably, in the stationary liquid of S2, the volume ratio of acetic acid, methanol and water is 11:11-33:1-2.

[0029] Preferably, in S4, after covering with 2-10 layers of paper towels, the cover glass is pressed firmly.

[0030] Preferably, in S5, the anhydrous ethanol is pre-cooled at -20°C.

[0031] Preferably, in S7, the step of dehydration with 60%-80% ethanol is repeated 2-4 times.

[0032] Preferably, in S10, the number of the probes is one or more than two, and each probe is 0.5 μM.

[0033] Preferably, the probe includes the 359 repeat probe shown in SEQ ID NO:1 and the rDNA probe shown in SEQ ID NO:2.

[0034] Preferably, in S14, the concentration of DAPI is 0.1-0.3 μg / mL, and the solvent is 2×SSC.

[0035] Preferably, the fixative solution in S2 and the 4% paraformaldehyde in S8 are both freshly prepared.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] The traditional method usually adopts a single fixation method of methanol glacial acetic acid to ensure a good chromosome expansion morphology, but it is not resistant to high temperatures and is often deformed at 70 degrees. At the same time, it is necessary to match a variable hybridization solution system, and it is still necessary to match a variable formamide rinsing system later. The experimental process is difficult to unify and the experimental results are unstable. If a single fixation method of 4% paraformaldehyde is used, it will be difficult to expand during the chromosome compression process, and it is difficult to obtain a good chromosome morphology, which is not suitable for use in chromosome compression. For this reason, the present invention further uses 4% paraformaldehyde treatment for double fixation on the basis of the methanol glacial acetic acid fixation of the chromosome compression, and it is found that this double fixation method can not only ensure that the chromosome has a good expansion morphology, but also enables it to resist high temperature deformation (up to 90 degrees or more), and further unifies the subsequent hybridization system and rinsing system, and does not need to explore various complex hybridization annealing processes and rinsing processes due to different probes, thereby constructing a universal, simple and efficient chromosome compression combined with in situ hybridization detection method. Compared with the traditional chromosome pressing combined with in situ hybridization technology, the present invention has higher clarity of chromosome structure morphology. While maintaining the hybridization signal, the chromosome break position can still be clearly observed in the non-signal area. In addition, it has a simpler operating process, more stable experimental results, and is convenient for novice operators. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a picture of chromosome pressing combined with in situ hybridization of Drosophila brain tissue; 1 and 2 in the picture represent two repeated experiments, the red light is the 359 probe, and the green light is the rDNA probe; the rDNA signal indicated by the white arrow is missing a piece compared with the adjacent homologous chromosome, indicating that the hybridization result is unstable; the red arrow indicates nonspecific binding; the white scale bar in the microscope picture represents 10μm.

[0039] Figure 2 This is a chromosome compression slide of Drosophila brain tissue combined with in situ hybridization, showing the chromosome structure and hybridization signals after treatment with different denaturation temperatures and times during hybridization; the red light is the 359 probe, and the green light is the rDNA probe; the white scale bar in the microscope image represents 10μm.

[0040] Figure 3 This is a picture of chromosome squashing combined with in situ hybridization of Drosophila brain tissue; 1 and 2 in the picture represent two repeated experiments, red light is 359 probe, and green light is rDNA probe; yellow arrows indicate chromosome breaks; the white scale bar in the microscope picture represents 10μm.

[0041] Figure 1-Figure 3 The white scale bar in the microscopic images represents 10 μm. DETAILED DESCRIPTION

[0042] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0043] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0044] Although chromosome pressing combined with in situ hybridization is the core technology of chromosome biology research, the operation process is cumbersome, and it is necessary to purchase an expensive hybridizer with high temperature accuracy. It also requires high technical experience of the operator, and different methods are used by different people. Even different probes often require different targeted experimental methods. The technical threshold is high, and the success rate of the experiment is not guaranteed. At the same time, there are generally two ways to fix the traditional chromosome pressing combined with in situ hybridization technology. One is methanol glacial acetic acid fixation. The chromosome morphology of this fixation method is good but it is not resistant to high temperatures and cannot form a clear image after in situ hybridization. At the same time, a denaturation temperature of 60-75 degrees is required, which brings trouble to the processing of nonspecific binding signals in the subsequent hybridization process and rinsing process. The other is 4% paraformaldehyde fixation, which cannot be unfolded during chromosome pressing, making it difficult to form a chromosome pressing with a good morphology. Furthermore, there is a lack of universal hybridization and rinsing systems that meet the needs of most probes (such as universal hybridization solution formulas and hybridization annealing conditions; universal rinsing solution formulas and rinsing temperature systems, etc.), which requires corresponding fine optimization and adjustment for each different probe. For novices, the threshold is high, the trial period is long, the experimental cost is high, and the difficulty is high. In addition, the current chromosome karyotype analysis combined with in situ hybridization technology can only be used in the interphase of cells, and can only detect aneuploidy of chromosomes 13, 18, 21, X, and Y. There is still a large gap in the application of other chromosome abnormalities. At the same time, for specific chromosome abnormalities, such as breakage translocations, the current detection methods based on chromosome karyotype analysis combined with in situ hybridization technology still have a lot of room for development.

[0045] In view of the problems of cumbersome operation, high difficulty in operation, unstable results, etc. in the traditional chromosome pressing film combined with in situ hybridization technology, the present invention aims to develop a simple and universal chromosome pressing film combined with in situ hybridization technology, so as to provide technical support for chromosome genetics research. First of all, in view of the problem that the traditional chromosome pressing film combined with in situ hybridization technology needs to use a complex hybridization system and a rinsing system, we consider that it may be affected by non-specific signals. Therefore, if the non-specific signal can be reduced or even eliminated as much as possible when the signal is added, it is possible to make the hybridization system and the rinsing system unified and universal. High temperature is the most effective way to solve non-specific signals. Just like PCR, if it can be denatured at more than 90 degrees and high temperature gradient annealing is performed from more than 90 degrees, it can be imagined that only probes that completely match the target will withstand such strict conditions. Secondly, considering that the chromosome pressing film combined with in situ hybridization technology needs to maintain the structure of the chromosome, and the traditional fixation method for maintaining the chromosome structure is not resistant to high temperature, the traditional hybridization system usually needs to use formamide to reduce the melting temperature (generally 60-70 degrees). For this reason, the present invention starts from improving the fixation method and seeks a fixation method that can withstand 90 degrees high temperature. Considering that different fixation methods have different principles and can tolerate different temperatures, the present invention explored a double fixation method and simplified the hybridization formula, retaining only 50% formamide, 10% dextran sulfate, and 2×SSC. The rinsing component was also simplified, retaining only the salt component 0.1×SSC, and using low-concentration salt to provide rinsing rigor. Finally, it was found through research that the concept of the present invention is very successful in terms of chromosome structure and signal clarity. The research content of the present invention is as follows:

[0046] In Example 1, the traditional methanol glacial acetic acid fixation method is used for fixation, and the fixation method completely loses the clear chromosome morphology at 70 degrees and 5 minutes. At the same time, it is found by using a simple rinsing process that if denaturation is performed at 70 degrees, on the one hand, the hybridization signal is unstable, and on the other hand, a large number of non-specific binding signals will be caused. The subsequent hybridization solution formula and rinse solution formula and temperature conditions must be matched according to different probes to reduce the non-specific signal. For this reason, the present invention further introduces a second-step fixation scheme, and uses a second fixing agent 4% paraformaldehyde for fixation. Because each reagent is fixed in a different way, the ability to resist high temperature can be different. As can be seen from Example 2, through the combination of these two fixation methods, even after undergoing a denaturation process of 90-95 degrees and 2-5 minutes, a clear chromosome morphology can still be maintained, and a strong and stable hybridization signal is provided. In addition, as can be seen from Example 3, since denaturation can be performed at high temperature, the non-specific binding of many probes is reduced, so the same hybridization solution formula and hybridization system can be used for any probe, without having to explore the annealing process of each probe. And the fact proves that, utilizing the hybridization solution system of the present invention, any probe only needs to be directly annealed to room temperature, and incubation in room temperature can realize the efficient hybridization of the probe. And only need to use the most general simplest rinse solution formula and room temperature rinse, just can realize the removal of non-specific signal. And, because the chromosome compression piece that the present invention sets up is combined with the in situ hybridization method chromosome structure clarity is high, thus after in situ hybridization, still can observe the breakage site on the chromosome. It can be seen that it has huge application potential in aspects such as detecting chromosome specific breakage translocation. Thus, the present invention sets up a kind of chromosome compression piece combined with in situ hybridization simple and general operation method, for chromosome behavior, genetics research and related detection provide technical support and reference.

[0047] In order to further clearly present the specific process of establishing the chromosome pressing combined with in situ hybridization detection method of the present invention, a detailed description is given below in conjunction with Examples 1-3.

[0048] Example 1-3 takes two probes as examples, one probe targets a very short repetitive sequence 359 repeat (only 5-10bp base repeats) in the heterochromatin region of the X chromosome in the brain tissue of Drosophila, and the other probe targets a highly transcribed and continuously repeated high-copy rDNA locus on the genome, demonstrating the process of constructing a simple operation method for chromosome compression combined with in situ hybridization in the present invention, providing a reference for technical development in related fields. The specific probes are as follows:

[0049] 359 repeat probe sequence (SEQ ID NO: 1):

[0050]

[0051] The 5' end of the probe is labeled with a Cy5 fluorescent signal (shown as red light in the figure).

[0052] rDNA probe sequence (SEQ ID NO:2):

[0053]

[0054] The 5' end of the probe is labeled with FITC fluorescent signal (shown as green light in the figure).

[0055] Both probes were synthesized by Agen Biotech and carry only a single fluorescent label.

[0056] In the following examples, the formula system of the 2×SSC buffer used is: 300 mM sodium chloride, 30 mM sodium citrate, the pH is adjusted to 7.0 with hydrochloric acid; 0.1×SSC is obtained by diluting 2×SSC.

[0057] Example 1: Methanol and glacial acetic acid fixation is not resistant to high temperatures

[0058] 1. Experimental operation

[0059] For the convenience of comparison with the subsequent examples, this example adopts the traditional methanol glacial acetic acid fixation method + optimized simple hybridization solution system and rinse solution system for operation, as follows:

[0060] (1) Select the third instar larvae of Drosophila melanogaster (purchased from the Drosophila Center of Tsinghua University) and dissect them in physiological saline to separate the brain tissue;

[0061] (2) Transfer the larval brain and immerse it in a drop (50 μL) of hypotonic buffer (0.5% sodium citrate dihydrate solution) at room temperature for 5 min.

[0062] (3) Transfer to a drop of fixative (50 μL, acetic acid: methanol: water = 11:11:2, v:v:v) and fix at room temperature for 10-20 seconds;

[0063] (4) Take a siliconized coverslip, add 2 μL of 45% glacial acetic acid to it, transfer the brain tissue into 45% glacial acetic acid, and cover it with a clean, non-siliconized slide;

[0064] (5) Cover with 2-3 layers of paper towels and press the coverslip firmly with your thumb to spread out the brain tissue and stretch out the chromosomes.

[0065] (6) After soaking in liquid nitrogen (until the boiling of liquid nitrogen stops), lift up the cover glass with a utility knife and soak the slide in anhydrous ethanol (stored at -20°C) for 15 minutes or overnight;

[0066] (7) Take out the slide and let it dry at room temperature;

[0067] (8) Wash the slides with 2× SSC buffer and dehydrate in 70% ethanol for 10 min. Repeat the dehydration twice, then treat in 95% ethanol for 5 min and air dry.

[0068] (9) Prepare hybridization solution according to the recipe: 50% formamide, 10% dextran sulfate, 2× SSC, 0.5 μM of each probe (359 repeat probe, rDNA probe), and finally add deionized water to 20 μL;

[0069] (10) Add hybridization solution to the sample and cover with a coverslip;

[0070] (11) Denaturation at 70°C for 5 min;

[0071] (12) Then quickly cool to room temperature and incubate in a humidified dark room at room temperature for 1 h;

[0072] (13) Wash three times with 0.1× SSC buffer, 15 min each time;

[0073] (14) The samples were stained with the nuclear dye DAPI (0.2 μg / mL, dissolved in 2×SSC) for 5 min;

[0074] (15) Quickly wash with 2× SSC and allow the slides to dry;

[0075] (16) The slides were mounted with Vectashield and analyzed using a microscope.

[0076] 2. Experimental results

[0077] Depend on Figure 1 It can be seen that the chromosomes fixed with methanol and glacial acetic acid can only resist the 70℃ denaturation temperature for 5 minutes in probe hybridization, which can lead to blurred chromosome morphology and structure. Figure 1 The hybridization signal of the rDNA (green light) locus indicated by the white arrow in the middle is unstable compared with the corresponding homologous chromosome (theoretically, the hybridization signal should be equivalent), which shows that the in situ hybridization system is not optimal. Figure 1 The red arrows in the middle show the non-specific binding signals of the two probes, which also proves that if the traditional methanol-glacial acetic acid fixation method and the hybridization denaturation temperature of 70°C are used, the matching hybridization solution formula and the rinse solution formula need to be explored later. Otherwise, the interference of non-specific binding signals cannot be eliminated.

[0078] Example 2: The double fixation method can resist deformation caused by high temperature (91-95 degrees), and the chromosome morphology is clear and the hybridization signal is normal

[0079] 1. Experimental operation:

[0080] (1) Select the third instar larvae of Drosophila melanogaster (purchased from the Drosophila Center of Tsinghua University) and put them into physiological saline for dissection to separate the brain tissue;

[0081] (2) Transfer the larval brain and immerse it in a drop (50 μL) of hypotonic buffer (0.5% sodium citrate dihydrate solution) at room temperature for 5 min.

[0082] (3) Transfer to a drop of fixative (50 μL, acetic acid: methanol: water = 11:11:2, v:v:v) and fix at room temperature for 10-20 seconds;

[0083] (4) Take a siliconized coverslip and add 2 μL of 45% glacial acetic acid to it. Transfer the brain tissue to 45% glacial acetic acid and cover it with a clean, non-siliconized slide.

[0084] (5) Cover with 2-3 layers of paper towels and press the coverslip firmly with your thumb to spread out the brain tissue and stretch out the chromosomes.

[0085] (6) After soaking in liquid nitrogen (until the boiling of liquid nitrogen stops), lift up the cover glass with a utility knife and soak the slide in anhydrous ethanol (stored at -20°C) for 15 minutes or overnight;

[0086] (7) Take out the slide and let it dry at room temperature;

[0087] (8) Wash the slides with 2× SSC and dehydrate in 70% ethanol for 10 min. Repeat the dehydration twice, then treat in 95% ethanol for 5 min and air dry.

[0088] (9) Prepare 4% paraformaldehyde (solvent is deionized water) and dehydrate the sample at room temperature for 30 min;

[0089] (10) Rinse the slides with PBS and dry them;

[0090] (11) Prepare hybridization solution according to the recipe: 50% formamide, 10% dextran sulfate, 2× SSC, 0.5 μM of each probe ((359 repeat probe, rDNA probe)), and finally add deionized water to 20 μL;

[0091] (12) Add hybridization solution to the sample and cover with a coverslip;

[0092] (13) The experimental treatments were denatured at 90°C for 2 min, 95°C for 2 min, and 95°C for 5 min;

[0093] (14) Then quickly cool to room temperature and incubate in a humidified dark room at room temperature for 1 h;

[0094] (15) Wash three times with 0.1× SSC, 15 min each time;

[0095] (16) The samples were stained with DAPI (0.2 μg / mL, dissolved in 2×SSC) for 5 min;

[0096] (17) Rapidly wash with 2× SSC and allow the slide to dry;

[0097] (18) The slides were mounted with Vectashield and analyzed using a microscope.

[0098] 2. Experimental results

[0099] Depend on Figure 2 It can be seen that the method developed in this embodiment adopts a double fixation method of methanol glacial acetic acid and 4% paraformaldehyde, which can make the chromosomes still maintain a clear chromosome structure morphology during high temperature denaturation at 90°C-95°C, 2min-5min. Among them, 90°C denaturation for 2 min, with the simple hybridization system and rinsing system of the present invention, although there are slight non-specific signals, both hybridization probes can produce strong and reliable hybridization signals. At the same time, such non-specific signals completely disappear when the denaturation temperature is increased to 95°C for 2 min, and both probes can produce strong and reliable hybridization signals, and after extending the denaturation time from 2min to 5min, clear chromosome morphology and strong hybridization signals can still be maintained. Compared with Example 1, it can be seen that the chromosome compression combined with in situ hybridization technology developed by the present invention has significant advantages.

[0100] Example 3: The double fixation method can clearly observe the break site on the chromosome structure while providing probe hybridization signals

[0101] 1. Experimental operation:

[0102] The 3rd instar larvae of Pol32 gene mutant fruit flies (purchased from the Drosophila Center of Tsinghua University) were collected for brain tissue dissection, because the mutant can cause chromosome breakage, and thus can be used as the tissue source for this embodiment. The hybridization denaturation temperature of this embodiment is 93 degrees, the time is 2 minutes, and the rest of the experimental operations are the same as Example 2.

[0103] 2. Experimental results: As shown in Example 1, the chromosome structure of the chromosomes produced by the traditional method combined with the in situ hybridization technique is fuzzy and cannot be further determined for the chromosome breakpoints. However, as shown in Example 2, the double fixation method of the present invention has a clear and clean chromosome morphology, which is expected to meet the accuracy of further determining chromosome breakpoints. This inference is further verified by this example. Figure 3 The yellow arrows in the figure indicate the breakpoints. Figure 3It can be seen that the chromosome breakage sites can be displayed in both the in situ hybridization signal map and the chromosome flattening morphology map, which further illustrates that the chromosome flattening combined with in situ hybridization technology developed by the present invention has significant advantages.

[0104] In summary, the present invention has established a universal and simple operation method for chromosome pressing combined with in situ hybridization. A double fixation method is used in the preparation of chromosome pressing, so that the chromosome can still maintain a clear chromosome structure at high temperature (90-95 degrees). At the same time, a universal hybridization system and rinsing system compatible with this have also been developed. There is no need to perform detailed optimization and matching exploration for each probe, no need to use expensive hybridization instruments, and no need for overnight hybridization, which is helpful for novice operation and conducive to meeting the processing requirements of multiple probes and multiple samples.

[0105] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

1. A simple operation method for chromosome pressing combined with in situ hybridization, characterized in that: The method is used for non-disease diagnosis purposes and comprises the following steps: S1. Treat the brain tissue with hypotonic buffer at room temperature for 3-10 minutes; S2, fixing with a fixing solution at room temperature for 10-20 seconds, wherein the fixing solution comprises acetic acid, methanol and water; S3, add 40%-50% acetic acid to the siliconized coverslip, transfer the brain tissue to 40%-50% acetic acid, and cover with a non-siliconized slide; S4. Press down the coverslip firmly to spread out the brain tissue and stretch out the chromosomes. S5. Treat the sample with liquid nitrogen, lift the cover glass, and place it in anhydrous ethanol for 10-30 minutes or overnight; S6. Take out the slide and dry it at room temperature; S7, wash the sample with 2×SSC, dehydrate in 60%-80% ethanol for 7-15 minutes, then treat in 90%-97% ethanol for 3-7 minutes, and dry; S8. Treat the sample with 4% paraformaldehyde at room temperature for 20-50 min; S9, rinse with PBS and dry; S10, adding hybridization solution to the sample and covering the cover glass, wherein the hybridization solution includes 50% formamide, 10% dextran sulfate, 2×SSC, and 0.5 μM probe; S11, denature at 90-95°C for 2-5 min; S12, then quickly cool to room temperature and incubate in a humidified dark environment at room temperature for 0.5-2 h or overnight; S13, wash with 0.1×SSC 2-5 times, 10-20 min each time; S14, stain with DAPI for 3-10 minutes; S15, quickly wash with 2×SSC and allow the slide to dry; S16. Cover the slides with Vectashield and analyze using a microscope.

2. The simple operation method of chromosome pressing combined with in situ hybridization according to claim 1, characterized in that: The hypotonic buffer described in S1 is 0.4%-0.6% sodium citrate dihydrate solution.

3. The simple operation method of chromosome pressing combined with in situ hybridization according to claim 1, characterized in that: In the stationary phase S2, the volume ratio of acetic acid, methanol and water is 11:11-33:1-2.

4. The simple operation method of chromosome pressing combined with in situ hybridization according to claim 1, characterized in that: In S4, cover with 2-10 layers of paper towels and then press down the coverslip firmly.

5. The simple operation method of chromosome pressing combined with in situ hybridization according to claim 1, characterized in that: In S5, the anhydrous ethanol is pre-cooled at -20°C.

6. The simple operation method of chromosome pressing combined with in situ hybridization according to claim 1, characterized in that: In S7, the step of dehydration with 60%-80% ethanol was repeated 2-4 times.

7. The simple operation method of chromosome pressing combined with in situ hybridization according to claim 1, characterized in that: In S10, the number of the probes is one or more than two, and each probe is 0.5 μM.

8. The simple operation method of chromosome pressing combined with in situ hybridization according to claim 7, characterized in that: The probes include the 359 repeat probe shown in SEQ ID NO:1 and the rDNA probe shown in SEQ ID NO:

2.

9. The simple operation method of chromosome pressing combined with in situ hybridization according to claim 1, characterized in that: In S14, the concentration of DAPI was 0.1-0.3 μg / mL, and the solvent was 2×SSC.

10. The simple operation method of chromosome pressing combined with in situ hybridization according to claim 1, characterized in that: The fixative described in S2 and the 4% paraformaldehyde described in S8 were freshly prepared.

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