High-sensitivity in-situ hybridization method

Abstract

According to the present invention, an in situ hybridization method is provided that is more sensitive and has fewer false positives than conventional methods. [Solution] A method for detecting a gene abnormality in which the structure of a chromosome is abnormal, comprising the steps of: labeling at least one nucleic acid of 10kb to 300kb; performing situ hybridization using the labeled nucleic acid in a buffer containing dextran sulfate with a molecular weight of 3,000 to 70,000 daltons; and detecting the hybridized nucleic acid.

Description

Technical Field

[0001] The present invention relates to an in situ hybridization method. More specifically, it relates to a highly sensitive in situ hybridization method.

Background Art

[0002] In the conventional in situ hybridization method (for example, Patent Document 1), there was a problem that false positives appeared at a certain rate in order to increase the length of the probe. In addition, since high molecular weight dextran was used, the viscosity of the hybridization buffer was high and there were problems in operation. Furthermore, when observing, it was necessary to observe with an oil immersion microscope, and there was also a problem that automation of detection was difficult.

[0003] Therefore, there has been a demand for a highly sensitive in situ hybridization method with fewer false positives and good operability.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] According to the present invention, an in situ hybridization method with higher sensitivity and fewer false positives than the conventional method is provided.

Means for Solving the Problems

[0006] (1) An in situ hybridization buffer containing at least one nucleic acid probe of 10 kb to 300 kb and dextran sulfate having a molecular weight of 3,000 to 70,000 daltons. Here, the nucleic acid probe is preferably a fluorescently labeled one. (2) The in situ hybridization buffer of (1), further comprising one or more selected from the group consisting of betaine, carnitine, and trimethylglycine. (3) The in situ hybridization buffer of (1), further containing polyethylene glycol. (4) A method for detecting a gene abnormality in which the structure of a chromosome is abnormal. A step of labeling at least one nucleic acid of 10kb to 300kb, The process involves performing in situ hybridization using the labeled nucleic acid in a buffer containing dextran sulfate with a molecular weight of 3,000 to 70,000 daltons, A step to detect hybridized nucleic acids, A method for detecting chromosomal structural abnormalities, comprising the characteristics of a method for detecting chromosomal structural abnormalities. (5) The method for detecting chromosomal structural abnormalities according to (4), wherein the buffer solution comprises one or more selected from the group consisting of betaine, carnitine, and trimethylglycine. (6) The method for detecting chromosomal structural abnormalities according to (4), wherein the buffer solution further contains polyethylene glycol. (7) A method for detecting off-target chromosomal structural abnormalities in genome editing, as described in (4). (8) A method for detecting chromosomal structural abnormalities as described in (4), which automatically detects chromosomal structural abnormalities. (9) A method for diagnosing a genetic abnormality in which the structure of a chromosome is abnormal. A step of labeling at least one nucleic acid of 10kb to 300kb, The process involves performing in situ hybridization using the labeled nucleic acid in a buffer containing dextran sulfate with a molecular weight of 3,000 to 70,000 daltons, A step to detect hybridized nucleic acids, A diagnostic method for chromosomal structural abnormalities, comprising the following characteristics. [Effects of the Invention]

[0007] According to the present invention, chromosomal abnormalities can be detected with higher sensitivity by using a shorter probe and a hybridization buffer containing low molecular weight dextran sulfate. [Brief explanation of the drawing]

[0008] [Figure 1] Figure 1 shows the location and length of the CCR5 gene probe. [Figure 2] Figure 2 shows photographs of cells with normal chromosomes (A), false-positive cells (B), and abnormal cells (C). [Figure 3] Figure 3 is a graph showing the relationship between probe length and the success rate (Figure 2A). [Figure 4] Figure 4 shows images of cells stained by the in-situ hybridization method of the present invention, using a 10x objective lens. [Figure 5] Figure 5 is a magnified view of the image within the white frame in Figure 4. [Figure 6] Figure 6 shows the results of image analysis of cells stained using the in-situ hybridization method of the present invention. [Figure 7] Figure 7 shows cells and gene detection using the Her2 gene probe. [Figure 8] Figure 8 shows the results of image analysis using the in-situ hybridization method of the present invention. [Modes for carrying out the invention]

[0009] As a result of diligent research, the inventors discovered that shortening the probe length reduces false positives, and that even with a shortened probe length, lowering the molecular weight of dextran sulfate ensures sufficiently strong fluorescence intensity, thus completing the present invention.

[0010] That is, according to one embodiment of the present invention, there is provided an in situ - hybridization buffer containing at least one nucleic acid probe of 10 kb to 300 kb and dextran sulfate having a molecular weight of 3,000 to 70,000 daltons. Further, the in situ - hybridization buffer of the present invention may further contain one or more selected from the group consisting of betaine, carnitine, and trimethylglycine, and may further contain polyethylene glycol.

[0011] The addition amount of betaine, carnitine, and trimethylglycine to the hybridization buffer of the present invention is not particularly limited as long as it promotes hybridization, but the preferable lower limit concentration is 10 μM or more, 100 μM or more, 1 mM or more, 10 mM or more, 50 mM or more, 100 mM or more, and the preferable upper limit concentration is 1000 mM or less, 500 mM or less, 300 mM or less.

[0012] By further adding polyethylene glycol to the hybridization buffer of the present invention, the hybridization rate can be further improved. The polyethylene glycol is not particularly limited as long as it promotes hybridization. For example, polyethylene glycol 200, polyethylene glycol 4000, polyethylene glycol 6000, polyethylene glycol 8000, etc. are preferably used. In this case, the preferable lower limit of the concentration of the added polyethylene glycol is 0.01% or more, 0.1% or more, 1% or more in weight% notation, and the preferable upper limit of the concentration is 10% or less, 5% or less, 2% or less. When adding polyethylene glycol to the hybridization buffer together with betaine, it is preferable to add both at the same concentration (weight%).

[0013] An example of the in situ - hybridization buffer of the present invention is shown below. Composition of in situ - hybridization solution (buffer) Dextran sulfate (molecular weight 3,000 - 70,000): 20 - 40% Betaine: 1 - 500 mM Polyethylene glycol (molecular weight 200 - 8,000): 1 - 500 mM Guanidine thiocyanate: 0.1 - 10% Formamide: 20 - 40% Sodium chloride: 100 - 800 mM Trisodium citrate dihydrate: 10 - 80 mM Tween - 20: 0.1% SDS: 0.1% Sodium deoxycholate: 0.1%

[0014] Conventionally, the length of the probe used for in situ hybridization has generally been as long as 300 kb - 800 kb. However, it has been found that when such a long probe is used, a certain degree of false positives occurs. For example, when the sequences on both sides of CCR5 as shown in Figure 1 are used as probes, if there is no chromosomal abnormality, hybridization occurs at adjacent positions, so the fluorescence of the two probes can be seen at almost the same position (Figure 2A). In contrast, in the case of chromosomal abnormalities, the fluorescence of each probe is observed at separated positions (Figure 2C). In the case of false positives, a pattern as shown in Figure 2B is exhibited. <00001​​​​​​​​​Next, while conventional hybridization buffers used dextran sulfate with a molecular weight of 500,000 daltons or more, this invention uses dextran sulfate with a molecular weight of 3,000 to 70,000 daltons. This reduces the viscosity of the in-situ hybridization buffer, making it easier to handle. Furthermore, lowering the molecular weight of dextran sulfate increases the fluorescence intensity, making fluorescence observation possible without microscopic observation using oil immersion. Image analysis is also possible with low-magnification objective lenses (e.g., 10×), allowing for the automatic observation of a large number of cells and the recording of image data. It is also possible to detect chromosomal abnormalities by visual inspection or automated scanning of this image data. To improve the accuracy of chromosomal abnormality detection, machine learning or deep learning using artificial intelligence may be used. In this case, the training data can be obtained by reading hybridization patterns of normal, false-positive, and abnormal cells. The preferred number of training data points is 100 or more, 1000 or more, 10,000 or more, 100,000 or more, and 1,000,000 or more, while the upper limits are 10,000,000 or less, 8,000,000 or less, 5,000,000 or less, and 3,000,000 or less.

[0017] Conventional methods require observation with an oil immersion objective lens of 60-100x, making it difficult to focus across the entire field of view. In this invention, in situ hybridization signals can be observed even with an objective lens of 10-40x, which is an unexpected benefit that opens the way to automating observation. The preferred lower limit of the objective lens magnification is 10x or higher, 20x or higher, 30x or higher, and 40x or higher, while the preferred upper limit of magnification is 60x or lower and 50x or lower.

[0018] In other words, according to another embodiment of the present invention, there is an invention for a method for detecting a gene abnormality in which the structure of a chromosome is abnormal, comprising the steps of: labeling at least one nucleic acid of 10kb to 300kb; performing in situ hybridization using the labeled nucleic acid in a buffer containing dextran sulfate with a molecular weight of 3,000 to 70,000 daltons; and detecting the hybridized nucleic acid. In this actual process, a washing step is included after hybridization, but for washing, the washing temperature and the concentration of SSC or SSPE can be appropriately selected as needed (References: Cancer Sci 105 (2014) 1212-1219, British Journal of Cancer (2012) 106, 727-732).

[0019] The preferred lengths of the nucleic acid probes used in the present invention are, at a lower limit, 10kb or more, 30kb or more, 50kb or more, and 70kb or more, and at a higher limit, 300kb or less, 250kb or less, 200kb or less, and 160kb or less.

[0020] However, in recent years, it has become technically possible to detect fluorescence from a single molecule, so it is also possible to perform in-situ hybridization using oligonucleotide probes. In this case, oligonucleotide probes with a length of about 20 bases may be used. The lengths of oligonucleotide probes in this case are 17 bases or more, 18 bases or more, 19 bases or more, and 20 bases or more, with the upper limit being the same as above. Multiple oligonucleotide probes may be designed, synthesized, and used. In the case of oligonucleotides, single-stranded DNA may be used when labeling the 5' end, or double-stranded DNA may be labeled and used as a probe. In the case of double-stranded DNA, the preferred lower limit of length is 17 bp or more, 18 bp or more, 19 bp or more, and 20 bp or more, with the upper limit being the same as above.

[0021] Performing in-situ hybridization using oligonucleotide probes requires highly sensitive detection equipment and specialized microscopy techniques, and the following equipment and techniques are commonly used.

[0022] a) TIRF (Total Internal Reflection Fluorescence) Microscope Features: TIRF microscopy is suitable for detecting single-molecule fluorescence signals by selectively exciting regions very close to the glass surface (usually below 100 nm), thereby reducing background noise. Applications: Used in experiments requiring a high signal-to-noise ratio for single-molecule detection, such as observing molecular dynamics on cell membranes.

[0023] i) Super-resolution microscopy techniques (STED, STORM, PALM) STED (Stimulated Emission Depletion) microscopy: This technique locally controls the fluorescence signal to achieve high resolution, enabling high-resolution detection of fluorescence from single molecules.

[0024] STORM (Stochastic Optical Reconstruction Microscopy) and PALM (Photo-Activated Localization Microscopy): These techniques allow for the precise identification of individual molecules by switching them on and off, enabling high-resolution imaging at the single-molecule level.

[0025] (c) Single-molecule fluorescence microscopy Features: Professional single-molecule fluorescence microscopy uses a combination of specific optical filters and a highly sensitive CCD (charge-coupled device) camera. This allows for the detection of weak signals from individual fluorescent molecules. Applications: Used to observe extremely fine biological processes, such as the dynamics of DNA and proteins, single-molecule interactions, and folding processes.

[0026] E) Light detection equipment High-sensitivity photodetectors: To detect the fluorescence of a single molecule, high-sensitivity photodetectors such as EM-CCDs (electron-multiplier CCDs) and sCMOS (scientific CMOS) cameras are necessary. These devices are suitable for observing single-molecule fluorescence because they can amplify and detect signals at very low light levels.

[0027] O) Optical systems and laser systems Precision Optical System: For single-molecule detection, the precision of the optical system is extremely important. High-quality objective lenses, precise filters, and a stable laser system are required. Low-noise lasers: Low-noise laser light sources are used to efficiently excite the fluorescence signal of a single molecule.

[0028] By appropriately combining these systems and devices, in-situ hybridization can be performed using oligonucleotide probes, and chromosomal abnormalities and off-target effects of genome editing can be detected by single-molecule imaging.

[0029] Furthermore, the preferred lower limit of the molecular weight of dextran sulfate contained in the in-situ hybridization buffer of the present invention is 3,000 daltons or more, 5,000 daltons or more, 6,000 daltons or more, 8,000 daltons or more, and 10,000 daltons or more, and the upper limit is 70,000 daltons or less, 65,000 daltons or less, 60,000 daltons or less, 50,000 daltons or less, 40,000 daltons or less, 30,000 daltons or less, and 20,000 daltons or less. In addition, the molecular weight range of dextran sulfate may be 3,000 daltons or more and less than 5,000 daltons or less or less than 6,000 daltons.

[0030] Figure 4 shows a photograph of cells stained by the in-situ hybridization method of the present invention, taken with a 10× objective lens. Fluorescence hybridized with the cells can be observed even with a 10× objective lens (Figure 5).

[0031] Even when using the Her2 gene as a probe, by magnifying the images captured with a 10x objective lens, it is possible to draw nuclear boundaries using DAPI-based image analysis to some extent (Figure 6). Furthermore, in Figure 7, hybridization signals can also be detected through image analysis. In Figure 8, the nucleus and hybridization signal can also be detected by image analysis.

[0032] Thus, by using the in-situ hybridization buffer and in-situ hybridization method of the present invention, sufficient fluorescence intensity can be obtained even with a short probe, eliminating the need for oil immersion observation, and enabling automated detection of cells, nuclei, and hybridization signals.

[0033] This means that chromosomal abnormalities can be detected automatically (by scanning) in a short time, the number of cells can be examined without limit, whether it's 1,000 or 10,000, and the image files can be saved to the electronic medical record.

[0034] Furthermore, the detection of chromosomal abnormalities can also be used to detect off-target mutations in genome editing. In genome editing, it is possible to edit genes in locations other than the intended target site (off-target mutation). In this case, chromosomal translocations or mutations in non-target sites occur. By scanning a large number of cells using the in-situ hybridization method of the present invention, off-target mutations can be detected, and cells containing unwanted mutations can be removed. [Examples]

[0035] (Example 1) Creation of a FISH probe The search for peripheral clone DNA containing the CCR5 gene region from a recombinant DNA library of human chromosomes was performed using the method described in Human BAC library: construction and rapid screening. Gene 1997; 191: p69-79. The clones searched are shown in Table 1. Each was fluorescently labeled with Texas Red (red) or FITC (green) using the Nick Translation Kit (GSP Institute / K009), suspended in the hybridization buffers listed below, and FISH probes No. 1 to No. 11 were prepared.

[0036] [Table 1]

[0037] Composition of in-situ hybridization solution (buffer) Dextran sulfate (molecular weight 3,000-70,000): 20-40% Betaine: 1-500mM Polyethylene glycol (molecular weight 200-8,000): 1-500 mM Guanidine thiocyanate: 0.1%~10% Formamide: 20-40% Sodium chloride: 100-800 mM Trisodium citrate dihydrate: 10-80 mM Tween -20: 0.1% SDS: 0.1% Sodium deoxycholate: 0.1%

[0038] After fixing the cells to glass slides, the cytoplasm was lysed and the nuclei were exposed using the Cell FISH Pretreatment Kit (GSP Institute / K002) to facilitate penetration of the FISH probe into the nucleus. The glass slides were immersed in a denaturing solution (70% formamide / 2×SSC) at 73-75°C for 5 minutes, and then dehydrated at room temperature with 70% and 100% ethanol for 1 minute each.

[0039] Fluorescently labeled FISH probes were denatured by heating 10 μl at 73-75°C for 5 minutes. The denatured FISH probes were dropped onto cells on a glass slide, the cells were covered with a coverslip, and the slides were placed on a hot plate at 45-50°C. The glass slides were then sealed with paper glue (KOKUYO) and left to stand overnight in a humidified chamber at 37°C. The coverslips were removed in 2×SSC / 0.3%NP-40 (room temperature), and after thorough washing in 2×SSC / 0.3%NP-40 (73°C) for 2 minutes, the slides were immersed in 2×SSC (room temperature) for 1 minute. After draining, the nuclei were counterstained with DAPI Countrestain 150 ng / ml (GSP Institute / K003). The signals were observed using a fluorescence microscope, and images were acquired using Keyence's image analysis software (BZ-H4A analysis application, Hybrid Cell Count).

[0040] Hybridization revealed both normal (Figure 2A) and false-positive (Figure 2B) results. In Figure 2A, the red and green fluorescence, which are normally adjacent, are both in almost the same position, but in Figure 2B, the red (R) and green (G) signals are in different positions. In Figure 2C (abnormal), the number of red (R) and green (G) signals is also different, with two red (R) signals and three green (G) signals.

[0041] Figure 3 shows the relationship between probe length and chromosomal normality. It can be seen that as the probe length decreases (as shown on the left of Figure 3, where the individual probe lengths are the same as in Figure 1), the normality rate (as shown in the graph on the right of Figure 3) increases. This is thought to be because shorter probe lengths reduce probe mismatches and thus lower the rate of false positives. In other words, shortening the probe length can improve the accuracy of in-situ hybridization.

[0042] (Example 2) FISH probes designed to detect the HER2 gene and the chromosome 17 marker (CEN17 / Patent No. 5554008) were prepared by labeling with TexasRed (red) or FITC (green) using the Nick Translation Kit (GSP Institute / K009) and then suspending them in hybridization buffer.

[0043] Formalin-fixed, paraffin-embedded xenografted breast cancer cell tissue (FFPE) sectioned to 5 μm thickness was mounted on glass slides to prepare tissue sections. The tissue sections were deparaffinized and pretreated with FFPE FISH Pretreatment Kit 2 (GSP Institute / K008). 10 μl of FISH probe was dropped onto the tissue sections on the glass slides, covered with coverslips, sealed with paper bond (KOKUYO), and placed on a 75°C hot plate for 5 minutes. Next, the sections were left to stand overnight in a humidified chamber at 37°C. The coverslips were removed by immersion in 2×SSC / 0.3%NP-40 (room temperature), and after thorough washing by immersion in 2×SSC / 0.3%NP-40 at 73°C for 2 minutes, the sections were immersed in 2×SSC at room temperature for 1 minute. After draining, the nuclei were counterstained with DAPI Countrestain 1500 ng / ml (GSP Institute / K004).

[0044] The signals were observed using a fluorescence microscope (ZEISS AXIO IMAGER A2), and images were acquired using a CCD camera (AXIOCAM506). Signal counting was also performed automatically using Keyence's image analysis software (BZ-H4A analysis application, hybrid cell count).

[0045] Conventionally, observations were made using a fluorescence microscope with a 100x or 60x oil immersion objective lens. However, by using the hybridization buffer and / or in-situ hybridization method of the present invention, observations can now be made with 40x, 20x, and 10x objective lenses without oil immersion (Figure 4). Furthermore, by capturing the microscope image with a CCD camera and magnifying the area within the white frame of the image (Figure 4), the fluorescence signals of the HER2 gene (red), CEN17 (green), and the nucleus (blue) could be clearly detected, confirming the amplification of the HER2 gene (Figure 5).

[0046] Because tissue specimens are thick, high-magnification oil immersion objectives cannot focus at a single point. However, as shown in Figure 4, ×40, ×20, and ×10 objectives were able to focus at a single point and capture a wide area at once. The image within the white frame was decomposed into RGB, and the image of the blue nucleus was automatically recognized. We succeeded in automatically recognizing the nucleus from that image. (Figure 6)

[0047] The image within the white frame was decomposed into RGB values, and the green CEN17 within the blue nucleus was automatically recognized (Figure 7), allowing us to quantify the number of CEN17 signals per nucleus (cell) (Figure 8).

[0048] In this project, we used Keyence's image analysis software (BZ-H4A analysis application, hybrid cell count). Because we were able to capture the FISH signal with a low-magnification objective lens, as demonstrated in this development, it will be possible in the future to automatically analyze the data by simply transferring the image to an analysis PC without the need for dedicated equipment. [Industrial applicability]

[0049] This invention can be used in the medical industry, for example, in plant breeding through genome editing. [Explanation of Symbols]

[0050] A Normal Pattern B False positive pattern C Abnormal Pattern G Green fluorescence R: Red fluorescence

Claims

1. An in-situ hybridization buffer comprising at least one 10kb–300kb nucleic acid probe and dextran sulfate with a molecular weight of 3,000–70,000 Daltons.

2. Furthermore, the in situ hybridization buffer according to claim 1 comprises one or more selected from the group consisting of betaine, carnitine, and trimethylglycine.

3. The in situ hybridization buffer according to claim 1, further comprising polyethylene glycol.

4. A method for detecting gene abnormalities in which the structure of chromosomes is abnormal. A step of labeling at least one nucleic acid of 10kb to 300kb, The process involves performing in situ hybridization using the labeled nucleic acid in a buffer solution containing dextran sulfate with a molecular weight of 3,000 to 70,000 daltons, A step to detect hybridized nucleic acids, A method for detecting chromosomal structural abnormalities, comprising the characteristics of a method for detecting chromosomal structural abnormalities.

5. The method for detecting chromosomal structural abnormalities according to claim 4, wherein the buffer solution comprises one or more selected from the group consisting of betaine, carnitine, and trimethylglycine.

6. The method for detecting chromosomal structural abnormalities according to claim 4, wherein the buffer solution further comprises polyethylene glycol.

7. A method for detecting chromosomal structural abnormalities according to claim 4, which detects off-target effects in genome editing.

8. A method for detecting chromosomal structural abnormalities according to claim 4, which automatically detects chromosomal structural abnormalities.

Images