Transposase-based probe as well as preparation method and application thereof

Abstract

The invention relates to a transposase-based probe as well as a preparation method and application thereof. Comprising the following steps: acquiring a target nucleic acid fragment; preparing a transposase compound, wherein the transposase compound comprises a joint with a label and transposase; and fragmenting the obtained target nucleic acid fragment by using the prepared transposase compound, and adding labeled linker sequences at the two ends of the fragmented nucleic acid sequence to obtain the probe. The obtained target nucleic acid fragment is high in specificity and amplification efficiency, plasmid does not need to be converted and extracted, the template obtaining way is simplified, and the resolution ratio of the hybridization detection method is improved. The nucleic acid template is broken into short segments by ingeniously utilizing the characteristic that the transposase randomly breaks a target nucleic acid segment, and compared with a long-segment probe, the probe with the short segments can reduce the difficulty of probe nucleic acid denaturation and the difficulty of sample hybridization, increase the fluorescence density in unit length and facilitate imaging.

Description

Technical Field

[0001] The present application relates to the field of molecular biology, and in particular to a transposase-based probe and a preparation method and application thereof. Background Art

[0002] In eukaryotic organisms, one-dimensional linear genomic DNA needs to be highly folded and compressed in the micrometer-level cell nucleus, forming a complex chromatin spatial conformation. DNA-FISH (DNA fluorescence in situ hybridization) technology can intuitively provide detailed information on the location and interaction of genes in cells, helping to understand gene regulation and biological functions.

[0003] There are two main methods for preparing traditional DNA-FISH probes. One method is to prepare DNA-FISH probes using a BAC clone of the target sequence as a template. The length of the BAC clone is generally between 100kb and 300kb. Through nick transfer, the labeled nucleotides are incorporated into the newly synthesized DNA chain to form a labeled probe, which is then used for DNA-FISH experiments; or the BAC clone DNA of the target sequence is cloned, and then the DNA template obtained by the BAC clone is cut into short fragments of 100bp to 300bp using DNA enzyme, and the ends are labeled with a labeling kit. Another method is to prepare Tn5-FISH probes using Tn5 transposase. The specific process is to first amplify the target genomic site by PCR, then use Tn5 transposase to fragment the amplified DNA, and finally perform a second round of PCR to amplify the fragments with labeled primers to form labeled probes. Finally, the prepared probes are used for DNA-FISH experiments. However, the above-mentioned traditional DNA-FISH methods have problems such as insufficient resolution and complicated probe preparation steps.

[0004] Therefore, the traditional technology still needs to be further improved. Summary of the invention

[0005] Based on this, the present application provides a method for preparing a transposase-based probe and its application, which improves the resolution of in situ hybridization detection and is highly efficient and specific.

[0006] The specific technical solutions are as follows:

[0007] In a first aspect of the present application, a method for preparing a transposase-based probe is provided, comprising the following steps:

[0008] obtaining a target nucleic acid fragment;

[0009] preparing a transposase complex, the transposase complex comprising a labeled adapter and a transposase;

[0010] The obtained target nucleic acid fragments are fragmented using the prepared transposase complex, and labeled adapter sequences are added to both ends of the fragmented nucleic acid sequences to obtain probes.

[0011] In the preparation method of the probe of the present application, the prepared transposase complex is used to fragment the target nucleic acid fragment obtained, and a labeled adapter sequence is added to both ends of the fragmented nucleic acid sequence to obtain a probe. Among them, for the obtained target nucleic acid fragment, the specificity is strong, the amplification efficiency is high, and no transformation and extraction of plasmids are required, which simplifies the template acquisition method and improves the resolution of the hybridization detection method. And the characteristic of the transposase randomly interrupting the target nucleic acid fragment is cleverly utilized to interrupt the nucleic acid template into shorter fragments. Compared with the probe of the long fragment, the probe of the shorter fragment can reduce the difficulty of the probe nucleic acid denaturation and the difficulty of hybridization with the sample, and increase the fluorescence density per unit length, which is conducive to imaging. When the transposase interrupts the template, the labeled adapter is added to the interrupted nucleic acid fragment to prepare the probe, while in the traditional Tn5-FISH method, the adapter of the transposon is not labeled, and the interrupted nucleic acid fragment needs to undergo a second round of PCR, and the label can be added to the nucleic acid fragment through the labeled primer to form a probe. Therefore, the method for preparing the probe of the present application is one-step, easy to operate, high in labeling efficiency, and reduces experimental costs.

[0012] In one embodiment, the transposase comprises Tn5 transposase.

[0013] In one embodiment, the target nucleic acid fragments include DNA and RNA.

[0014] In one embodiment, the label is selected from one or more of a radioactive label, a fluorescent group label, a colorimetric label, a quantum dot label and a biotin label;

[0015] Optionally, the fluorescent group in the fluorescent group label is selected from one of FAM, HEX, VIC, CY5, ROX, TexsaRed and Quasar705, and the fluorescent groups for different target nucleic acid fragments are different.

[0016] In one embodiment, the step of preparing the transposase complex comprises the following steps:

[0017] Synthesizing a first primer, a second primer and a third primer; the first primer and the second primer carry the label respectively; wherein the nucleotide sequence at the 3' end of the first primer is complementary to the nucleotide sequence of the third primer, and the nucleotide sequence at the 3' end of the second primer is complementary to the nucleotide sequence of the third primer;

[0018] Annealing the synthesized labeled first primer and the labeled second primer with the third primer to form a first adapter having a double-stranded structure and a second adapter having a double-stranded structure;

[0019] The first linker containing a double-stranded structure, the second linker containing a double-stranded structure and a naked transposase enzyme are mixed to assemble into a transposase complex.

[0020] In one embodiment, the sequence of the first primer is shown as SEQ ID No: 3;

[0021] And / or, the sequence of the second primer is shown as SEQ ID No: 4;

[0022] And / or, the sequence of the third primer is as shown in SEQ ID No: 5.

[0023] In one embodiment, the molar ratio of the labeled first primer to the third primer is 1-1.5:1;

[0024] And / or, the molar ratio of the labeled second primer to the third primer is 1-1.5:1.

[0025] In one embodiment, the annealing treatment procedure includes: heating at 94°C~98°C for 1min~3min; cooling to 72°C~78°C at 0.01°C / s~0.2°C / s, and incubating for 1min~3min; then cooling to 62°C~68°C at 0.01°C / s~0.2°C / s, and incubating for 1min~3min; then cooling to 47°C~53°C at 0.01°C / s~0.2°C / s, and incubating for 1min~3min; then cooling to 34°C~40°C at 0.01°C / s~0.2°C / s, and incubating for 1min~3min; then cooling to 18°C~22°C at 0.01°C / s~0.2°C / s, and incubating for 1min~3min; then cooling to 2°C~6°C at 0.01°C / s~0.2°C / s, and incubating for 1min~3min.

[0026] In a second aspect of the present application, a hybridization probe is also provided, which is prepared using the above-mentioned method for preparing a transposase-based probe.

[0027] The third aspect of the present application also provides a hybridization detection kit, which contains the hybridization probe as described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the process of Tn5-labeled DNA-FISH in Example 1;

[0029] Figure 2The figure is a result of nucleic acid capillary electrophoresis analysis of the PCR amplification product of genomic DNA in Example 1;

[0030] Figure 3 The figure is the result of nucleic acid capillary electrophoresis on the labeled linker in Example 1;

[0031] Figure 4 This is a diagram showing the results of nucleic acid capillary electrophoresis analysis of the fragments after Tn5 transposition in Example 1;

[0032] Figure 5 This is the imaging image of Tn5-labeled DNA-FISH detection in Example 1;

[0033] Figure 6 This is a diagram showing the imaging analysis results of Tn5-labeled DNA-FISH detection in Example 1. DETAILED DESCRIPTION

[0034] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail below, and the preferred embodiments of the present application are given. In the following description, many specific details are set forth to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present application, so the present application is not limited by the specific embodiments disclosed below.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present application belongs. The terms used herein in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The term "and / or" used herein includes any and all combinations of one or more related listed items. The reagents or instruments used herein without indicating the manufacturer are conventional products that can be purchased.

[0036] In this application, the concentration values ​​involved include fluctuations within a certain range. For example, fluctuations can be allowed within the corresponding accuracy range. For example, 2%, fluctuations within ±0.1% are allowed. For values ​​that are large or do not require too fine control, larger fluctuations are also allowed. For example, 100mM, fluctuations within the range of ±1%, ±2%, ±5%, etc. are allowed. In relation to molecular weight, fluctuations within ±10% are allowed.

[0037] In this application, m / v means mass-to-volume ratio, and %(m / v) means the mass percentage contained in a certain volume of a mixed system. For example, if the concentration of substance A in a mixed system is 5%(m / v), it means that 5 grams of substance A is contained in every 100 ml of the mixed system.

[0038] In this application, the terms "first", "second", etc. in "first primer", "second primer", etc. are only used for distinguishing descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", etc. only serve the purpose of non-exhaustive enumeration description and should be understood not to constitute a closed limitation on quantity.

[0039] The term "primer" refers to an oligonucleotide, whether naturally present in a purified restriction digest or produced synthetically, which can act as a starting point for synthesis when placed under conditions that induce the synthesis of a primer extension product complementary to a nucleic acid strand (e.g., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH). The primer is preferably single-stranded for maximum efficiency in amplification, but may alternatively be double-stranded. If double-stranded, the primer is first treated to separate its chains before being used to prepare an extension product. Preferably, the primer is an oligodeoxyribonucleotide. The primer should be long enough to initiate the synthesis of an extension product in the presence of an inducing agent. The exact length of the primer will depend on many factors, including temperature, primer source, and the use of the method. For example, in some embodiments, primers range from 10-100 or more nucleotides (e.g., 10-300, 15-250, 15-200, 15-150, 15-100, 15-90, 20-80, 20-70, 20-60, 20-50 nucleotides, etc.).

[0040] In this application, μmol / L and μM both represent micromoles per liter and can be used interchangeably. mmol / L and mM both represent millimoles per liter and can be used interchangeably. mol / L and M both represent moles per liter and can be used interchangeably.

[0041] The term "nucleic acid" refers to any molecule comprising nucleic acid, including but not limited to DNA or RNA.

[0042] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0043] As shown in the background technology, the traditional DNA-FISH probe preparation is prepared using BAC clones. The length of BAC clones is usually between 100kb and 300kb. Through nick transfer, the labeled nucleotides are incorporated into the newly synthesized DNA chain to form a labeled probe. The nick transfer method relies on the existing BAC clones, and the amount of DNA required is relatively large. The ratio of enzyme usage needs to be optimized, and the size of the generated fragments cannot be precisely controlled. In addition, the basic unit of the three-dimensional structure of chromatin, the topologically associated domain (TAD), ranges from 0.2Mb to 1Mb, and the length of the chromatin loop related to gene expression regulation is at the kb level. The resolution of the probe prepared by BAC clones is not enough. Therefore, the preparation process of traditional DNA-FISH probes based on BAC clones is cumbersome and costly, and there is a problem of insufficient resolution when labeling genomic interactions less than 500 kb. Another traditional Tn5-FISH probe preparation is performed using Tn5 transposase. The specific process is to first amplify the target genomic site through PCR, then use Tn5 transposon to fragment the amplified DNA, and finally perform a second round of PCR to amplify the fragments with labeled primers to form labeled probes. Finally, the prepared probe is used for DNA-FISH experiments. The genomic region prepared by this method can be less than 1 kb, but the preparation of the probe requires a second round of PCR, which has many steps and is more complicated.

[0044] Based on this, the present application provides a method for preparing a transposase-based probe, comprising steps S10 to S30.

[0045] Step S10: Obtain target nucleic acid fragments.

[0046] In some embodiments, obtaining the target nucleic acid fragment comprises the following steps:

[0047] A nucleic acid sample is taken, and a PCR amplification reaction is performed using amplification primers of the target nucleic acid fragment to prepare the target nucleic acid fragment.

[0048] In some embodiments, the target nucleic acid fragments include DNA and RNA.

[0049] In some embodiments, the target nucleic acid fragments can be derived from any sample containing the target nucleic acid.

[0050] In some embodiments, the above-mentioned samples include cells, tissues or living bodies, or samples or cultures obtained from any source, and biological samples. Biological samples can be obtained from animals (including humans) and refer to biological materials or compositions found therein, including but not limited to bone marrow, blood, serum, platelets, plasma, interstitial fluid, urine, cerebrospinal fluid, nucleic acids, DNA and tissues.

[0051] In a specific example, the sample is derived from Drosophila S2 cells.

[0052] Step S20: preparing a transposase complex, wherein the transposase complex comprises a labeled linker and a transposase.

[0053] In some embodiments, the transposase comprises Tn5 transposase.

[0054] Furthermore, the above-mentioned Tn5 transposase includes one or more of BGI Tn5 transposase, BeyoNGS™ Tn5 transposase, and NovoNGS®Tn5 Transposase.

[0055] In some embodiments, the label is selected from one or more of a radioactive label, a fluorescent group label, a colorimetric label, a quantum dot label and a biotin label.

[0056] In a specific example, the label is a fluorescent group label.

[0057] In some embodiments, the fluorescent group is selected from one of FAM, HEX, VIC, CY5, ROX, Texsa Red and Quasar 705, and the fluorescent groups for different target nucleic acid fragments are different.

[0058] Specifically, the step of preparing the transposase complex in step S20 includes steps S21 to S23.

[0059] Step S21: synthesizing a first primer, a second primer and a third primer; the first primer and the second primer carry the same and / or different labels respectively; wherein the nucleotide sequence at the 3' end of the first primer is complementary to the nucleotide sequence of the third primer; and the nucleotide sequence at the 3' end of the second primer is complementary to the nucleotide sequence of the third primer.

[0060] Optionally, the 15-25 nucleotide sequence at the 3' end of the first primer is complementary to the nucleotide sequence of the third primer; the 15-25 nucleotide sequence at the 3' end of the second primer is complementary to the nucleotide sequence of the third primer.

[0061] In a specific example, the 19 nucleotide sequence at the 3' end of the first primer is complementary to the 19 nucleotide sequence of the third primer, and the 19 nucleotide sequence at the 3' end of the second primer is also complementary to the 19 nucleotide sequence of the third primer.

[0062] In some embodiments, the sequence of the first primer is as shown in SEQ ID No: 3. Specifically, the nucleotide sequence as shown in SEQ ID No: 3 is: 5'- / Cy5.5 / TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3'.

[0063] In some embodiments, the sequence of the second primer is as shown in SEQ ID No: 4. Specifically, the nucleotide sequence as shown in SEQ ID No: 4 is: 5'- / Cy5.5 / GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3'.

[0064] In some embodiments, the sequence of the third primer is as shown in SEQ ID No: 5. Specifically, the nucleotide sequence as shown in SEQ ID No: 5 is: 5'- / Phos / CTGTCTCTTATACACATCT-3'.

[0065] Step S22: The synthesized labeled first primer and the labeled second primer are annealed with the third primer to form a first linker having a double-stranded structure and a second linker having a double-stranded structure, respectively.

[0066] In some embodiments, the molar ratio of the labeled first primer to the third primer is 1-1.5:1, for example, 1.1:1, 1.2:1, 1.3:1, 1.4:1 or 1.5:1, or a range consisting of any two of these values.

[0067] In some embodiments, the molar ratio of the labeled second primer to the third primer is 1-1.5:1, for example, 1.1:1, 1.2:1, 1.3:1, 1.4:1 or 1.5:1, or a range consisting of any two of these values.

[0068] In some embodiments, the annealing treatment procedure includes: heating at 94°C~98°C for 1min~3min; cooling to 72°C~78°C at 0.01°C / s~0.2°C / s, and incubating for 1min~3min; then cooling to 62°C~68°C at 0.01°C / s~0.2°C / s, and incubating for 1min~3min; then cooling to 47°C~53°C at 0.01°C / s~0.2°C / s, and incubating for 1min~3min; then cooling to 34°C~40°C at 0.01°C / s~0.2°C / s, and incubating for 1min~3min; then cooling to 18°C~22°C at 0.01°C / s~0.2°C / s, and incubating for 1min~3min; then cooling to 2°C~6°C at 0.01°C / s~0.2°C / s, and incubating for 1min~3min.

[0069] In a specific example, the annealing procedure includes: heating at 95°C for 2 minutes; cooling to 75°C at 0.1°C / s and incubating for 2 minutes; cooling to 65°C at 0.1°C / s and incubating for 2 minutes; cooling to 50°C at 0.1°C / s and incubating for 2 minutes; cooling to 37°C at 0.1°C / s and incubating for 2 minutes; cooling to 20°C at 0.1°C / s and incubating for 2 minutes; cooling to 4°C at 0.1°C / s and incubating for 2 minutes.

[0070] Step S23: Mix the first linker containing a double-stranded structure, the second linker containing a double-stranded structure and the naked transposase enzyme to assemble into a transposase complex.

[0071] Step S30: fragmenting the obtained target nucleic acid fragments using the prepared transposase complex, and adding labeled linker sequences to both ends of the fragmented nucleic acid sequences to obtain probes.

[0072] In the preparation method of the probe of the present application, the prepared transposase complex is used to fragment the obtained target nucleic acid fragment, and a labeled adapter sequence is added to both ends of the fragmented nucleic acid sequence to obtain a probe. Among them, for the obtained target nucleic acid fragment, the specificity is strong, the amplification efficiency is high, and no transformation and extraction of plasmids are required, which simplifies the template acquisition method and improves the resolution of the hybridization detection method. And the characteristic of the transposase randomly interrupting the target nucleic acid fragment is cleverly utilized to interrupt the nucleic acid template into shorter fragments. Compared with the probe of the long fragment, the probe of the shorter fragment can reduce the difficulty of the probe nucleic acid denaturation and the difficulty of hybridization with the sample, and increase the fluorescence density per unit length, which is conducive to imaging. When the transposase interrupts the template, the labeled adapter is added to the interrupted nucleic acid fragment to prepare the probe, while in the traditional Tn5-FISH method, the adapter of the transposon is not labeled, and the interrupted nucleic acid fragment needs to undergo a second round of PCR, and the labeled primer can be added to the nucleic acid fragment to form a probe. Therefore, the method for preparing the probe of the present application is one-step, easy to operate, high in labeling efficiency, and reduces experimental costs.

[0073] One embodiment of the present application further provides a hybridization probe, which is prepared using the above-mentioned transposase-based probe preparation method.

[0074] The hybridization probes used in hybridization detection can significantly improve the resolution. The hybridization probes can effectively verify chromatin interactions, especially interactions within specific genomic regions, and provide new tools and possibilities for studying genome structure.

[0075] In one embodiment of the present application, a hybridization detection kit is also provided, comprising the above-mentioned hybridization probe.

[0076] The hybridization detection kit has high detection resolution and strong specificity.

[0077] The following is a detailed description in conjunction with specific examples. The following examples do not include other components except for inevitable impurities unless otherwise specified. The reagents and instruments used in the examples are conventionally selected in the art unless otherwise specified. The experimental methods for which specific conditions are not specified in the examples are implemented according to conventional conditions, such as the conditions described in the literature, books, or methods recommended by the manufacturer.

[0078] Example 1

[0079] 1. Experimental steps

[0080] (1) Extraction of cell genomic DNA

[0081] According to the cell type to be subjected to DNA-FISH experiment, 10 million freshly cultured cells are taken, for example, genomic DNA is extracted from Drosophila S2 cells, and genomic DNA is extracted using a universal genomic extraction kit according to the instructions.

[0082] (2) Amplification of target sequence

[0083] a) Design specific primers for the target sequence, use genomic DNA as a template, and prepare the PCR reaction system shown in Table 3 below on ice. Taking the amplification of the l(1)G0020 gene on the X chromosome of Drosophila as an example, extract the genomic DNA from the above Drosophila S2 cells as a template for the PCR reaction, and perform PCR using primers that specifically amplify the l(1)G0020 gene. The PCR primer sequences are shown in Figure 1 Specifically, the forward primer sequence is: 5'-TTCTCCAGGAATCCCAGATG-3' (SEQ ID No: 1); the reverse primer sequence is: 5'-CGTAAATCTGCCCGAGGATA-3' (SEQ ID No: 2).

[0084] Table 1

[0085]

[0086] b) Set the PCR program shown in Table 2 below, and place the prepared PCR reaction system in a PCR instrument for reaction.

[0087] Table 2

[0088]

[0089] c) Purify the PCR product using the Zymo DNA purification kit (Zymo Research, Cat# D4013) according to the instructions.

[0090] d) The purified amplification products were used to measure the DNA concentration using Qubit double-stranded DNA quantitative reagent (Thermo Fisher, Cat#Q32851) according to the instructions; the nucleic acid capillary electrophoresis instrument Qsep100 (BIOptic) was used to analyze the DNA length distribution according to the instructions. The amplification products were stored at -20°C for future use.

[0091] (3) Preparation of transposase complex

[0092] a) Add 1×TNE buffer and transfer the synthesized labeled (e.g. Cy5.5) ME_A / B primers (primer sequences see Figure 1 ) and ME primers (primer sequences see Figure 1 ) dry powder was dissolved into 100 μM, mixed at a volume ratio of 1:1 and 1.1:1, and annealed to form adapters (adaptor A / B (50 μM)) according to the procedure shown in Table 3 below (adaptor sequences are shown in Figure 1 ). Specifically, the nucleotide sequence of the first primer (ME_A) is: 5'- / Cy5.5 / TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3' (SEQ ID No: 3); the nucleotide sequence of the second primer (ME_B) is: 5'- / Cy5.5 / GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3' (SEQID No: 4); the nucleotide sequence of the third primer (ME) is 5'- / Phos / CTGTCTCTTATACACATCT-3' (SEQ ID No: 5); the nucleotide sequence of the first adapter (adaptor A) is: 5' - / Cy5.5 / TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3' 3'-TCTACACATATTCTCTGTC / Phos / -5' (SEQ ID No: 6); the second adapter (adaptor B) The nucleotide sequence is: 5'- / Cy5.5 / GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3' 3'-TCTACACATATTCTCTGTC / Phos / -5' (SEQ ID No: 7).

[0093] Adaptor A / B used Qubit double-stranded DNA quantification reagent (Thermo Fisher, Cat# Q32851) to measure DNA concentration according to the instructions; nucleic acid electrophoresis was performed using 4-20% TBE gradient gel (Thermo Fisher, Cat# EC6225BOX) to analyze annealing. Adaptor A / B was kept at -20℃ for future use.

[0094] Table 3

[0095]

[0096] b) Prepare and thoroughly mix the following Tn5 assembly system shown in Table 4 on ice:

[0097] Table 4

[0098]

[0099] The prepared reaction system was placed at room temperature for 1 hour to complete the assembly of the Tn5 transposon complex, and then placed at -20°C for later use.

[0100] (4) Preparation of probe (protect from light)

[0101] a) Prepare and thoroughly mix the Tn5 transposition system shown in Table 5 below on ice (DNA is the target sequence to be amplified; the amount of Tn5 used can be adjusted according to the length of the fragments after fragmentation. The more enzyme is used, the shorter the fragments after enzyme digestion, generally 140 bp).

[0102] Table 5

[0103]

[0104] b) Set up the program shown in Table 6 below and place the prepared Tn5 reaction system in a PCR instrument for reaction.

[0105] Table 6

[0106]

[0107] c) Reaction termination and digestion: Add 50 µL ChIP Elution Buffer (final SDS concentration is 0.5%) and 1 µL proteinase K to the above reaction system and react at 65°C for 30 min.

[0108] d) Purify the probe using the Zymo DNA purification kit (Zymo Research, Cat# D4013) according to the instructions.

[0109] e) The purified probe was used to measure the DNA concentration using Qubit double-stranded DNA quantitative reagent (Thermo Fisher, Cat#Q32851) according to the instructions; the nucleic acid capillary electrophoresis instrument Qsep100 (BIOptic) was used to analyze the DNA length distribution according to the instructions. The probe was placed at -20 °C for future use.

[0110] (5) Cell patch

[0111] a) Slide preparation: Take out a 6-well plate, place the slide in the well, wash the cover slide twice with 1 mL of pure ethanol, and dry at 37°C for 1 hour; cover the slide with 100 μL of poly-lysine (Sigma, Cat# P4707) and leave at room temperature for 20 minutes; remove the poly-lysine and dry at room temperature for 1 hour; wash once with 2 mL of DPBS and dry at room temperature for 1 hour. Plant the cells to be subjected to DNA-FISH on the slide treated with poly-lysine.

[0112] b) Seeding cells: Take freshly cultured cells and dilute them to a certain concentration with preheated complete culture medium so that the density of cells after seeding on the slide is about 60%. Add the diluted cells to the slide and culture them for 20 minutes under the corresponding cell culture conditions. Remove the culture medium and wash twice with DPBS.

[0113] (6) Cell fixation: Add 2 mL 4% PFA and fix at room temperature for 10 min. Remove the supernatant and wash three times with DPBS.

[0114] (7) Cell permeabilization: Add 2 mL of 0.5% Triton X-100 / DPBS and permeabilize for 5 min at room temperature. Remove the supernatant and wash once with DPBS.

[0115] (8) Removal of RNA: Add 2 mL of 100 µg / mL RNase A / DPBS and incubate at 37 °C for 1 h. Remove the supernatant and wash three times with DPBS.

[0116] (9) Liquid nitrogen treatment: Add 2 mL of 20% glycerol / DPBS and incubate at room temperature for 30 min. Immerse the slide in liquid nitrogen for 30 s, wait for the slide to thaw and then place it in 20% glycerol / DPBS; repeat the liquid nitrogen treatment.

[0117] (10) Hydrochloric acid treatment: Add 2 mL of 0.1 M HCl to treat the cells and incubate at room temperature for 5 min. Remove the supernatant and wash three times with 2× SSC.

[0118] (11) Denaturation of probes and cell samples

[0119] a) Add 2 mL 2×SSC / 50% formamide to treat the cells and incubate at room temperature for 30 min.

[0120] b) Take 5 ng of probe and add it to 110 µL of hybridization buffer. Incubate at 80°C in the dark for 10 min for heat denaturation.

[0121] c) Quickly cover the cell side of the slide with the probe and heat-denature it at 80°C for 5 min.

[0122] (12) Hybridization of probe and cell samples (protected from light): Transfer the hybridization device of probe and cells to 42°C and incubate overnight. Wash three times with 2×SSC, three times with 0.1×SSC, and twice with DPBS. Add 2 mL of DPBS and store at 4°C protected from light.

[0123] (13) Imaging: The sample is placed under a laser confocal or super-resolution microscope for imaging observation.

[0124] 2. Results

[0125] (1) Results of target sequence amplification: Taking the amplification of the l(1)G0020 gene on the Drosophila X chromosome as an example, first, genomic DNA was extracted from Drosophila S2 cells, and then used as a template for the PCR reaction. PCR was performed using primers that specifically amplify the l(1)G0020 gene. The fragments of the amplified product were analyzed using a nucleic acid capillary electrophoresis instrument Qsep100.

[0126] The results are as follows Figure 2 As shown, it can be seen that the amplified product is single and the fragment lengths are consistent, indicating that this method efficiently and specifically amplifies a template that can be used for probe preparation.

[0127] (2) Adaptor A / B annealing results: When the molar ratio of ME_A / B primer with fluorescent label (Cy5.5) and ME primer is 1:1, the gradient nucleic acid electrophoresis (as shown below) Figure 3 As shown in A), it can be seen that after annealing, there are two bands in the adapter (Adaptor A / B) lanes. The molecular weight of the brighter band is obviously larger than that of the ME_A / B primer and the ME primer before annealing, but there is a weaker band below the band with a molecular weight close to that of the ME primer. This result shows that under this annealing molar ratio, the two primers can anneal to form an adapter, but there is still some ME primer remaining. In order to achieve the ideal annealing result, the amount of ME_A / B primer needs to be increased. Next, the molar ratio of the ME_A / B primer with fluorescent label (Cy5.5) and the ME primer was increased to 1.1:1, and the gradient nucleic acid electrophoresis (as shown below) was used. Figure 3 As shown in B), it can be seen that the molecular weight of the band in the adapter (Adaptor A / B) lane after annealing is significantly larger than the primer band before annealing, and there are no other miscellaneous bands below the band. This result shows that the 1.1:1 annealing ratio and gradient annealing program are very suitable for annealing of the labeled Tn5 adapter, which can make the primers of the upper and lower chains of the adapter combine well through annealing, and no original primers remain.

[0128] After the primer denaturation at 95°C, the Adaptor A / B gradient annealing program sets a uniform gradient cooling program of 75°C, 65°C, 50°C, 37°C, 20°C, and finally 4°C, and an incubation program at the corresponding temperature, so that the primers can fully anneal and hybridize to form stable hybrid products after denaturation. In previous literature, the Tn5 adaptor annealing program only has a uniform gradient cooling program after denaturation, and no additional incubation time is set at different temperature sections. In this way, the Tn5 transposon assembled by the adaptor formed by annealing will have more primer residues in the product after transposition. The residue will affect the subsequent efficiency.

[0129] (3) Results of probe preparation: Taking the l(1)G0020 gene locus on the Drosophila X chromosome as the DNA-FISH target locus, the gene sequence amplified by PCR was used as a template and the assembled Tn5 transposon with a label (Cy5.5) was used to fragment it. At the same time, the labeled adaptor was transposed to the fragment of the target sequence to form a labeled probe. The fragments after Tn5 transposition were analyzed using the nucleic acid capillary electrophoresis instrument Qsep100 (as shown below). Figure 4 As shown in the figure, it can be seen that the template is broken into diffuse fragments ranging from 20 to 1000 bp by Tn5 transposase, with a peak value of about 140 bp. These fragments are shorter than the probes prepared by the BAC cloning method and have higher resolution, so they can be used as probes for DNA-FISH.

[0130] (4) Results of Tn5-labeled DNA-FISH imaging: Using Drosophila Kc167 cells (female, XX) and Drosophila S2 cells (male, XY) as experimental materials, and the l(1)G0020 gene site on the Drosophila X chromosome as the DNA-FISH target site, DNA-FISH experiments were performed on the two cell lines using labeled probes prepared with Tn5-labeled transposons. The imaging results are as follows (see below) Figure 5 The left image shows two fluorescent spots in female Kc167 cells (XX), corresponding to the l(1)G0020 gene locus on the two X chromosomes of females, while the right image shows a larger single fluorescent spot in male S2 cells (XY), corresponding to the l(1)G0020 gene locus on the one X chromosome of males. Figure 6 As shown in the middle four figures, these foci are resolved into many small dots arranged in a ring structure; quantitative analysis shows (as shown below Figure 6 The circumference of the transcription loop in male cells is significantly larger, which supports the hypothesis that genes on the X chromosome are hyperactivated in males due to more open chromatin. This result shows that the probe prepared by this method can efficiently and specifically hybridize with the target gene in situ in cells.

[0131] The above results show that this method can be used to prepare DNA-FISH probes conveniently, quickly and efficiently. It is a novel and economical technology that can significantly improve the resolution. Tn5-labeled DNA-FISH can effectively detect chromatin interactions, especially interactions within specific genomic regions, providing a new tool for studying genome structure.

[0132] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0133] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solutions of the present application in detail, but it cannot be understood as limiting the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several modifications and improvements can be made, which all belong to the scope of protection of the present application. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent of this application shall be based on the contents of the attached claims, and the description and drawings can be used to explain the contents of the claims.

Claims

1. A method for preparing a transposase-based probe, characterized in that: The steps include: obtaining a target nucleic acid fragment; preparing a transposase complex, the transposase complex comprising a labeled adapter and a transposase; The obtained target nucleic acid fragments are fragmented by using the prepared transposase complex, and labeled adapter sequences are added to both ends of the fragmented nucleic acid sequences to obtain probes.

2. The method for preparing a transposase-based probe according to claim 1, wherein: The transposase comprises Tn5 transposase.

3. The method for preparing a transposase-based probe according to claim 1, wherein: The target nucleic acid fragments include DNA and RNA.

4. The method for preparing a transposase-based probe according to claim 1, wherein: The label is selected from one or more of radioactive label, fluorescent group label, colorimetric label, quantum dot label and biotin label; Optionally, the fluorescent group in the fluorescent group label is selected from one of FAM, HEX, VIC, CY5, ROX, Texsa Red and Quasar 705, and the fluorescent groups for different target nucleic acid fragments are different.

5. The method for preparing a transposase-based probe according to any one of claims 1 to 4, characterized in that: The step of preparing the transposase complex comprises the following steps: Synthesizing a first primer, a second primer and a third primer; the first primer and the second primer carry the label respectively; wherein the nucleotide sequence at the 3' end of the first primer is complementary to the nucleotide sequence of the third primer, and the nucleotide sequence at the 3' end of the second primer is complementary to the nucleotide sequence of the third primer; Annealing the synthesized labeled first primer and the labeled second primer with the third primer to form a first adapter having a double-stranded structure and a second adapter having a double-stranded structure; The first linker containing a double-stranded structure, the second linker containing a double-stranded structure and a naked transposase enzyme are mixed to assemble into a transposase complex.

6. The method for preparing a transposase-based probe according to claim 5, characterized in that: The sequence of the first primer is shown in SEQ ID No: 3; And / or, the sequence of the second primer is as shown in SEQ ID No:4; And / or, the sequence of the third primer is as shown in SEQ ID No:

5.

7. The method for preparing a transposase-based probe according to claim 6, wherein: The molar ratio of the labeled first primer to the third primer is 1-1.5:1; And / or, the molar ratio of the labeled second primer to the third primer is 1-1.5:

1.

8. The method for preparing a transposase-based probe according to any one of claims 6 to 7, characterized in that: The annealing treatment procedure includes: heating at 94°C-98°C for 1min-3min; cooling to 72°C-78°C at 0.01°C / s-0.2°C / s, and incubating for 1min-3min; then cooling to 62°C-68°C at 0.01°C / s-0.2°C / s, and incubating for 1min-3min; then cooling to 47°C-53°C at 0.01°C / s-0.2°C / s, and incubating for 1min-3min; then cooling to 34°C-40°C at 0.01°C / s-0.2°C / s, and incubating for 1min-3min; then cooling to 18°C-22°C at 0.01°C / s-0.2°C / s, and incubating for 1min-3min; then cooling to 2°C-6°C at 0.01°C / s-0.2°C / s, and incubating for 1min-3min.

9. A hybridization probe, characterized in that The probe is prepared by the method for preparing a transposase-based probe according to any one of claims 1 to 8.

10. A hybridization detection kit, characterized in that: Contains the hybridization probe as claimed in claim 9.

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