Signal amplification probe set, application thereof and in-situ hybridization detection kit

Abstract

The present application relates to a signal amplification probe set and its application and an in-situ hybridization detection kit, and belongs to the technical field of molecular biology. The signal amplification probe set comprises a specific capture probe pair and a specific modification probe for recognizing target nucleic acid. The specific capture probe pair comprises a linker sequence with a restriction endonuclease NdeI recognition sequence in reverse complement. When the specific capture probe pair recognizes the target nucleic acid, the linker sequence is paired in reverse complement to form a T-shaped structure. The specific modification probe is modified on a 5'-TAAGCCCCATATGCGTTCAGCTAGTACGCATATGGGGCT-3' sequence. Under the action of NdeI endonuclease and DNA ligase, the signal amplification probe set can quickly realize specific binding of the target sequence and amplification of the hybridization signal, and obtain information of multiple chromosomes (or chromosome fragments) or multiple gene states in cells.

Description

Technical Field

[0001] This invention relates to a signal amplification probe set and its application, as well as an in situ hybridization detection kit, belonging to the field of molecular biology technology. Background Technology

[0002] In situ hybridization (ISH) is a technique that utilizes complementary base sequences between single strands of nucleic acid molecules. A specifically labeled nucleic acid with a known sequence is used as a probe to hybridize with nucleic acids in cells or tissue sections. The location of the target nucleic acid on the tissue, cell, or chromosome is then revealed using specific detection methods. This method has high sensitivity and specificity, allowing for further exploration of cellular functional expression and its regulatory mechanisms at the molecular level. It has become an important tool in modern cell biology and molecular biology research. Currently, ISH detection probes are typically prepared by inserting fluorescein or haptens using PCR or synthetic modification. However, conventional PCR has a low insertion rate, and synthetic modification is costly, failing to yield high-quality probes. Furthermore, conventional signal amplification methods require multiple hybridization and incubation steps, which are cumbersome and time-consuming. Summary of the Invention

[0003] To address the aforementioned problems, the first objective of this invention is to provide a signal amplification probe set that has low synthesis and modification costs, and can achieve signal amplification simply by modifying the probe with specific parameters, without requiring multiple incubation steps, making it simple and convenient.

[0004] A second objective of this invention is to provide a specific modified probe.

[0005] The third objective of this invention is to provide an in situ hybridization detection kit.

[0006] The fourth objective of this invention is to provide the application of signal amplification probe sets or in situ hybridization detection kits in tissue in situ hybridization detection.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A signal amplification probe set includes specific capture probe pairs and specific modification probes for recognizing target nucleic acids;

[0009] The specific capture probe pair includes a first probe and a second probe. The first probe includes a first specific recognition sequence and a 3' adapter sequence, and the second probe includes a second specific recognition sequence and a 5' adapter sequence. The 3' and 5' adapter sequences are inversely complementary. When recognizing the target nucleic acid, the first and second probes bind to the target nucleic acid through the specific recognition sequence, and the 3' and 5' adapter sequences pair inversely complementary pairs to form a T-shaped structure. The 3' and 5' adapter sequences contain a restriction endonuclease NdeI recognition sequence.

[0010] The specific modification probe is obtained by modifying the base sequence, and the nucleotide sequence of the base sequence is: 5'-TAAGCCC CATATG CGTTCAGCTAGTACG CATATG GGGCT-3'; the modification includes phosphorylation modification of the 5' end and fluorescence modification or hapten modification of the first G base at the 5' end.

[0011] This invention introduces the restriction endonuclease NdeI recognition sequence into the working probe set, which has low synthesis and modification costs. This method only requires one modified probe, and the rest are all conventional primer sequences. Moreover, a large number of probes can be produced by synthesizing the probe and sequence in one step. The operation is simple and only requires two steps to complete the signal amplification operation.

[0012] Preferably, the 3' end connector sequence is: 5-AGCTTCGA CATATG ATCATCG-3'; the 5' end connector sequence is 5'-CGATGAT CATATG TCGAAGCT-3'.

[0013] The CA↓TATG sequence in the adapter sequence is the recognition sequence of the restriction endonuclease NdeI. The cleavage site is shown by the arrow. Each pair of probes can specifically bind to a specific segment of the target gene and form a T-shaped structure (T-type probe). The binding sites at the ends of the two probes can be recognized and cleaved by the NdeI endonuclease, forming a 5' protruding TA sticky end.

[0014] Preferably, the specific recognition sequence on the first probe and the second probe include a second specific recognition sequence of the same length.

[0015] The use of specific recognition sequences of the same length ensures that the binding capacity at both ends is equal, preventing situations where one end can bind while the other cannot during hybridization, which could lead to experimental failure.

[0016] More preferably, the length of the first specific recognition sequence and the second specific recognition sequence is 20 bp.

[0017] The preferred length of the specific recognition sequence is 20 bp, which can be adjusted between 18-30 bp. The shorter the length, the weaker the binding ability and the lower the binding efficiency during hybridization. Excessive length will greatly increase the cost.

[0018] Preferably, the number of specific capture probe pairs is ≥2.

[0019] A specific modification probe, wherein the specific modification probe is obtained by modifying a base sequence, the nucleotide sequence of which is: 5'-TAAGCCC CATATG CGTTCAGCTAGTACG CATATG GGGCT-3'; the modification includes phosphorylation modification of the 5' end and fluorescence modification or hapten modification of the first G base at the 5' end.

[0020] The specific modification probes of this invention are universal probes targeting specific capture probe pairs, and can be used for different target genes, reducing the cost of synthesis and modification. After denaturation at 94°C for 3 minutes and cooling at room temperature for 20 minutes, the specific modification probes can form hairpin or central protrusion structures, such as... Figure 1 As shown, the long arm of the secondary structure has a TA sticky end that can bind to the end of the T-type probe. Due to the characteristics of the NdeI endonuclease, when the number of protected bases at both ends of the recognition site is less than 7 bp, the cleavage efficiency of the endonuclease is 0. Therefore, the NdeI enzyme can only cleave the modified probe after it binds to the T-type probe. So when the modified probe is linked to the T probe, the endonuclease cleaves, exposing the TA end, which can then bind to the next modified probe. When the cleaved circular sequence binds to the T probe, the cleavage site is preserved, and the endonuclease can cleave it again. However, when the long arm of the modified probe binds to the T probe, the cleavage site disappears, and the endonuclease cannot recognize it, thus preserving its long chain state.

[0021] An in situ hybridization detection kit containing a signal amplification probe set.

[0022] The in situ hybridization detection kit of the present invention includes a signal amplification probe set and a detection reagent. Under the action of NdeI endonuclease and DNA ligase, the specific binding of the target sequence and the amplification of the hybridization signal are rapidly achieved. After DAPI counterstaining, the signals of multiple colored probes hybridized to the DNA target sequence in the cell nucleus can be rapidly observed and analyzed under a fluorescence microscope to obtain information on the status of multiple chromosomes (or chromosome fragments) or multiple genes in the cell.

[0023] Preferably, the method includes a hybridization buffer and a probe dilution solution; the hybridization buffer comprises the following components: 30% (v / v) formamide, 28% (v / v) dextran sulfate, 2× sodium citrate buffer, and 1M guanidine isothiocyanate; the probe dilution solution comprises the following components: 0.5 U / ul NdeI restriction endonuclease, 10xH Buffer 10% (v / v), DNA ligase 0.5 U / ul, and 10x DNA Ligase Buffer 10% (v / v).

[0024] Application of signal amplification probe sets or in situ hybridization detection kits in tissue in situ hybridization detection.

[0025] The signal amplification probe set or in situ hybridization detection kit of the present invention can be applied to in situ detection of tissue DNA and in situ detection of tissue RNA, without the need for multiple hybridization and incubation steps, and the operation steps are simple and convenient.

[0026] Preferably, the probe is first incubated with a specific capture probe, and then a second hybridization is performed using a denatured specific modified probe, wherein the denaturation is carried out at 94°C for 3 minutes and then cooled.

[0027] The specific modified probe needs to be denatured at 94℃ for 3 min and cooled at room temperature for 20 min before use, and then diluted with probe diluent to a final concentration of 8 pmol / L. Attached Figure Description

[0028] Figure 1 This is a diagram of the secondary structure formed by the specifically modified probe of this invention;

[0029] Figure 2 This is a schematic diagram illustrating the working principle of the signal amplification probe group of the present invention.

[0030] Figure 3 This is a HER2 in situ hybridization detection image on breast tissue in Example 4 of the present invention. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments, experimental examples and comparative examples are all commercially available.

[0032] The following is a brief introduction to some of the biological materials, experimental reagents, and experimental equipment involved in the following examples and experimental cases:

[0033] Reagent Information

[0034] Hybridization buffer: 30% (v / v) formamide, 28% (v / v) dextran sulfate, 2×SSC (sodium citrate buffer), 1M guanidine isothiocyanate, in water.

[0035] Probe dilution buffer: NdeI restriction endonuclease 0.5 U / ul, 10x H Buffer 10% (V / V), DNA Ligase 0.5 U / ul, 10x DNA Ligase Buffer 10% (V / V), solvent is water.

[0036] The pepsin treatment solution, washing solution 1, washing solution 2 and DAPI counterstaining solution were from Henan Sainote Biotechnology Co., Ltd., product number: CF4001 (Fluorescence In Situ Hybridization Sample Pretreatment Kit).

[0037] Example 1: A signal amplification probe assembly

[0038] The signal amplification probe set in this embodiment includes specific capture probe pairs and specific modification probes for recognizing target nucleic acids. Taking the human epidermal growth factor receptor 2 gene (HER2) as an example, the technical solution in this embodiment of the invention will be clearly and completely described.

[0039] 1. Design and fabrication of specific capture probes

[0040] (1) Download the full-length sequence of the human epidermal growth factor receptor 2 (HER2) gene from the NCBi Genbank database.

[0041] (2) The full-length genome sequence obtained in step (1) is used to design oligonucleotide fragments with a length of 40 bp using Oligominer software.

[0042] (3) The oligonucleotide fragments obtained in step (2) were subjected to BLAST specificity analysis one by one, and 215 specific probe sequences were screened out.

[0043] (4) Divide the specific oligonucleotide fragment obtained in step (3) into two segments, namely the first probe (first 20 bp) and the second probe (last 20 bp). Add an adapter sequence AGCTTCGA to the 3' end of the first probe. CATATG ATCATCG; Add the connector sequence CGATGAT to the 5' end of the second probe. CATATG TCGAAGCT yielded the HER2-specific capture probe sequences shown in Table 1.

[0044] Table 1. HER2-specific capture probe sequence list

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051] (5) The specific probe sequence obtained in step (4) is used by Sangon Biotech (Shanghai) Co., Ltd. to synthesize oligonucleotide probes, with each probe having a concentration of 10 pmol / L.

[0052] Note: In step 5, the CA↓TATG sequence in the adapter sequence is the NdeI restriction endonuclease recognition sequence, and the cleavage site is shown by the arrow; each pair of probes can specifically bind to a specific segment of the HER2 gene and form a T-shaped structure. The binding sites at the ends of the two probes can be recognized and cleaved by the NdeI endonuclease, forming 5' protruding TA sticky ends.

[0053] (6) Mix the probes obtained in step (5) at a ratio of first probe: second probe (v / v) = 1:1, and dilute with hybridization buffer to 100 times to obtain the specific capture probe solution.

[0054] Note: The specific capture probe solution obtained in step (6) is the working solution of the capture probe and can be prepared before use.

[0055] 2. Design and preparation of specifically modified probes

[0056] (1) Preferably, a specific sequence containing two restriction endonuclease NdeI recognition sequences is used:

[0057] 5-TAAGCCC CATATG CGTTCAGCTAGTACG CATATG GGGCT-3'

[0058] (2) Phosphorylation modification at the 5' end, and cy3 fluorescence modification at the first G base at the 5' end; after probe synthesis, add water to dissolve the powdered probe according to the instructions.

[0059] (3) The modified probe obtained in step (2) is denatured at 94°C for 3 min, cooled at room temperature for 20 min, and diluted with probe diluent to a final concentration of 8 pmol / L to obtain the specific modified probe solution.

[0060] Note: Specifically modified probes, after denaturation at 94℃ for 3 minutes and cooling at 50℃ for 20 minutes, can form hairpin or central protrusion structures, such as... Figure 1As shown, the long arm of the secondary structure has a TA sticky end that can bind to the end of the T-type probe. Due to the characteristics of the NdeI endonuclease, when the number of protected bases at both ends of the recognition site is less than 7 bp, the cleavage efficiency of the endonuclease is 0. Therefore, the NdeI enzyme can only cleave the modified probe after it binds to the T-type probe. So when the modified probe is linked to the T probe, the endonuclease cleaves it, exposing the TA end, which can then bind to the next modified probe. When the cleaved circular sequence binds to the T probe, the cleavage site is preserved, and the endonuclease can cleave it again. However, when the long arm of the modified probe binds to the T probe, the resulting sequence is CATAAG. This sequence cannot be recognized by NdeI, the cleavage site disappears, and the endonuclease cannot recognize it, thus preserving its long chain state. Figure 2 As shown.

[0061] Example 2: A Specifically Modified Probe

[0062] The specific design and preparation method of the modified probe in this embodiment are as described in step 2 of embodiment 1.

[0063] Example 3: An in situ hybridization detection kit

[0064] The in situ hybridization detection kit of this embodiment includes a signal amplification probe set, hybridization buffer, probe dilution solution, pepsin treatment solution, washing solution 1, washing solution 2 and DAPI counterstaining solution.

[0065] Example 4: Application of signal amplification probe sets or in situ hybridization detection kits in tissue in situ hybridization detection technology.

[0066] This embodiment applies the signal amplification probe set or in situ hybridization detection kit to tissue in situ hybridization detection technology. The HER2 signal amplification probe set prepared in Example 1 is used to perform in situ hybridization detection of HER2 in breast tissue. The specific implementation method is as follows:

[0067] 1. Tissue section pretreatment

[0068] 1.1 Bake the slices at around 56-65℃ for at least three hours (overnight).

[0069] 1.2 Preheat the pepsin treatment solution to 37°C and keep it warm for later use.

[0070] 1.3 Place the sections in xylene at room temperature for 10 minutes, and repeat this step twice.

[0071] 1.4 Place the sections in anhydrous ethanol at room temperature for 5 minutes, and repeat this step once.

[0072] 1.5 Treat the sections with 85% ethanol and 70% ethanol at room temperature for 3 minutes each time.

[0073] 1.6 Rinse with deionized water for 2 minutes, repeat 3 times.

[0074] 1.7 Boil an appropriate amount of pretreatment buffer solution in a microwave oven, keep it warm, and then place the slide in the microwave for 20 minutes for repair.

[0075] Note: Antigen retrieval can also be performed by adding an appropriate amount of pretreatment buffer to a pressure cooker and autoclaving for 5 minutes.

[0076] 1.8 Remove the slides and wash them three times with deionized water. Place the slides directly into the protease treatment solution and digest for 15 minutes.

[0077] Note: Digestion time varies depending on the type of tissue (cell) and the thickness of the slice. Generally speaking, it is recommended to conduct preliminary experiments to find the optimal digestion time for the sample.

[0078] 1.9 Rinse with deionized water for 2 minutes, three times.

[0079] 1.10 Dehydration: Treat in 70%, 85%, and 100% ethanol solutions for 2 minutes each, then air dry.

[0080] 2. Degeneration and hybridization

[0081] 2.1 Remove the specific capture probe solution from the refrigerator, centrifuge briefly, and use a pipette to add 10 μl to the tissue area.

[0082] 2.2 Cover the sample with a coverslip, avoiding air bubbles during the process, and then seal the slide with mounting adhesive.

[0083] 2.3 Place the slices in a hybridization apparatus, denature at 90℃ (±2℃) for 2 minutes, and hybridize at 55℃ for 60 minutes.

[0084] 3. Post-hybridization treatment

[0085] 3.1 Heat the washing solution I to 73±1℃ in a water bath 30 minutes in advance, and carefully remove the sealing adhesive.

[0086] 3.2 Place the slide in Washing Solution II for 5-10 minutes, then remove the coverslip.

[0087] 3.3 Place the slide in washing solution I for 2 minutes, during which time lift the slide once to ensure thorough washing.

[0088] 3.4 Place the slices in Washing Solution II and let stand for 2 minutes.

[0089] Note: Washing solution I and washing solution II are mainly used to wash away excess unhybridized sequences to prevent them from affecting downstream experiments.

[0090] 3.5 Dehydration: Treat in 70%, 85%, and 100% ethanol solutions for 2 minutes each, then air dry.

[0091] 4. Secondary hybridization

[0092] 4.1 Remove the specific modified probe solution from the refrigerator, centrifuge briefly, and use a pipette to add 20 μl to the tissue area.

[0093] 4.2 Cover the sample with a coverslip to avoid air bubbles; seal the slide with sealing adhesive.

[0094] 4.3 Place the slices in a hybridization apparatus and incubate at 37°C for 30-60 minutes.

[0095] 5. Post-hybridization treatment

[0096] 5.1 Heat the washing solution I to 73±1℃ in a water bath 30 minutes in advance, and carefully remove the sealing adhesive.

[0097] 5.2 Place the slide in Washing Solution II for 5-10 minutes, then remove the coverslip.

[0098] 5.3 Place the slide in washing solution I for 2 minutes, during which time lift the slide once to ensure thorough washing.

[0099] 5.4 Place the slices in Wash Buffer II and let stand for 2 minutes.

[0100] 5.5 Dehydration: Treat in 70%, 85%, and 100% ethanol solutions for 2 minutes each, then air dry.

[0101] 6. Observation

[0102] 6.1 Add 10 μl of DAPI counterstain to the tissue section, cover with a coverslip to avoid air bubbles, and incubate in the dark at -20°C for 15 minutes.

[0103] 6.2 Observe and count under a fluorescence microscope.

[0104] The results are as follows Figure 3 As shown, the signal points are clearly visible. Normally, there are two copies of the HER2 gene in each cell, so two signal points will appear. However, since it is a tissue section, some cells may be incomplete, resulting in one signal or no signal, which is normal.

[0105] In summary, the signal amplification probe set of the present invention is a newly developed signal amplification method that can produce clear and distinct signal points in tissue in situ hybridization detection and can be applied to routine probe production and clinical testing.

[0106] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A signal amplification probe assembly, characterized in that: This includes specific capture probe pairs and specific modified probes for recognizing target nucleic acids; The specific capture probe pair includes a first probe and a second probe. The first probe includes a first specific recognition sequence and a 3' adapter sequence, and the second probe includes a second specific recognition sequence and a 5' adapter sequence. The 3' and 5' adapter sequences are inversely complementary. When recognizing the target nucleic acid, the first and second probes bind to the target nucleic acid through the specific recognition sequence, and the 3' and 5' adapter sequences pair inversely complementary ways to form a T-shaped structure. The 3' and 5' adapter sequences contain a restriction endonuclease NdeI recognition sequence. The 3' adapter sequence is: 5'-AGCTTCGA CATATG ATCATCG-3'; the 5' end connector sequence is 5'-CGATGAT CATATG TCGAAGCT-3'; The specific modification probe is obtained by modifying the base sequence, and the nucleotide sequence of the base sequence is: 5'-TAAGCCC CATATG CGTTCAGCTAGTACG CATATG GGGCT-3'; the modification includes phosphorylation at the 5' end and fluorescence modification of the first G base at the 5' end.

2. The signal amplification probe assembly according to claim 1, characterized in that: The first probe has a specific recognition sequence and the second probe has a second specific recognition sequence of the same length.

3. The signal amplification probe assembly according to claim 2, characterized in that: The lengths of the first and second specific recognition sequences are 20 bp.

4. The signal amplification probe assembly according to claim 1, characterized in that: The number of specific capture probe pairs is ≥2.

5. An in situ hybridization detection kit comprising the signal amplification probe set of claim 1.

6. The in situ hybridization detection kit according to claim 5, characterized in that: The in situ hybridization detection kit includes a hybridization buffer for specific capture probe pairs and a probe dilution solution for specific modified probes. The hybridization buffer comprises the following components: 30% (v / v) formamide, 28% (v / v) dextran sulfate, 2× sodium citrate buffer, and 1M guanidine isothiocyanate. The probe dilution solution comprises the following components: 0.5 U / μL NdeI restriction endonuclease, 10x H Buffer 10% (v / v), 0.5 U / μL DNA ligase, and 10x DNA Ligase Buffer 10% (v / v).

7. The application of the signal amplification probe set as described in any one of claims 1 to 4 in in situ hybridization detection in tissues, characterized in that: The application is for non-disease diagnosis purposes.

8. The application of the method described in claim 7 in tissue in situ hybridization detection, characterized in that: First, the probe was incubated with a specific capture probe, and then a second hybridization was performed using a denatured specific modified probe. The denaturation was carried out at 94°C for 3 minutes and then cooled.

9. The application of the in situ hybridization detection kit as described in claim 5 or 6 in tissue in situ hybridization detection, characterized in that: The application is for non-disease diagnosis purposes.

10. The application of the method described in claim 9 in tissue in situ hybridization detection, characterized in that: First, the probe was incubated with a specific capture probe, and then a second hybridization was performed using a denatured specific modified probe. The denaturation was carried out at 94°C for 3 minutes and then cooled.

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