A capture probe targeting a pig whole genome promoter long-range interaction site, a kit and application thereof

Abstract

The application belongs to the technical field of biotechnology and three-dimensional genomics, and discloses a capture probe for targeting a promoter long-range interaction site of a whole pig genome, a kit and application thereof. The technology can efficiently enrich and accurately detect the regulatory elements in the pig genome which have long-range interaction with the promoter, significantly improves the feasibility and reliability of the long-range interaction research of the pig genome, and provides important technical support for the functional research of the pig genome, the genetic mechanism analysis of complex traits, and the innovation of molecular breeding technology.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and three-dimensional genomics, specifically relating to a capture probe, kit, and application of a target porcine whole-genome promoter remote interaction site. Background Technology

[0002] With the continuous innovation and development of three-dimensional genomics technology and the successful completion of the Encyclopedia of DNA Elements (ENCODE) project, the complex mechanisms of genome regulation have been elucidated in greater depth. Numerous studies have confirmed that various regulatory elements in the genome (such as enhancers and silencers) do not function confined to neighboring gene regions, but rather often regulate the transcription process of target genes across linear distances of the genome through long-range chromatin interactions, thereby precisely regulating the organism's phenotypic characteristics and physiological functions. This discovery provides an important theoretical foundation for elucidating gene expression regulatory networks and uncovering the molecular mechanisms associated with complex traits, and has also promoted the development of technical methods for specifically capturing long-range genomic interaction fragments.

[0003] Currently, technologies for studying three-dimensional long-range interactions in the genome have diversified, with techniques such as Hi-C, ChIA-PET, ChIA-ATAC, and Hi-ChIP emerging and being applied. However, these existing technologies still have significant limitations when applied to the study of three-dimensional interactions in the pig genome: on the one hand, they are constrained by both data quality and detection resolution, making it difficult to accurately capture the specific long-range interaction relationships between promoters and regulatory elements in the pig genome; on the other hand, the experimental costs of such technologies are high, facing a significant economic feasibility bottleneck in large-scale pig genome functional studies and breeding-related applications, thus restricting their promotion and application in the field of pig genetics and breeding.

[0004] In contrast, Capture Hi-C technology stands out due to its unique advantages: through a targeted capture strategy mediated by specific probes, it can accurately enrich long-range interaction fragments in target regions (such as promoter regions) across the entire genome. This not only significantly improves the resolution and specificity of interaction detection but also significantly reduces experimental costs, effectively overcoming the aforementioned shortcomings of traditional three-dimensional genomics techniques. However, to date, no specific capture probes for detecting long-range interactions between promoters and regulatory elements in the entire porcine genome have been publicly reported, and there is a lack of dedicated tools that can be directly used for three-dimensional interaction studies in the porcine genome, severely hindering in-depth research on the functional analysis of the porcine genome and related genetic and breeding molecular mechanisms. Summary of the Invention

[0005] To address the aforementioned technical problems, the purpose of this invention is to provide a capture probe, kit, and applications targeting remote interaction sites of the pig genome promoter. This technology belongs to the interdisciplinary field of biotechnology and three-dimensional genomics. Its successful development not only fills the technological gap in pig-specific Capture Hi-C probes, providing an efficient, precise, and economical technical means for three-dimensional interaction research of the pig genome, but also provides important support for functional analysis of the pig genome, the discovery of genetic mechanisms of complex traits, and innovation in molecular breeding technologies, possessing significant academic value and broad application prospects.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A capture probe targeting remote interaction sites of promoters across the entire porcine genome, wherein the capture probe is two specific probes designed for each promoter region of the entire porcine genome, and the nucleotide sequence of the probe is shown in SEQ ID NO. 1-600; The GC content of the capture probe sequence is 20-60%; Each of the aforementioned capture probes is 120 bp in length.

[0007] Preferably, the capture probe is located near the GATC restriction site in the promoter region of the whole pig genome.

[0008] Preferably, the capture probe further includes a modifying group; The modifying group is located at the 5' or 3' end of the probe and includes one or more of biotin, fluorescein, carboxyl modifying group, thiol modifying group and phosphorylation modifying group.

[0009] An application of a capture probe in the construction of a high-throughput targeted capture library includes the following steps: (1) Select fresh or liquid nitrogen-preserved pig tissues and grind them into powder in liquid nitrogen. Then, use a formaldehyde solution with a mass concentration of 1-2% to cross-link the tissue samples. After the cross-linking is completed, add a de-cross-linking agent to terminate the cross-linking and collect the cross-linked tissue samples after treatment. (2) Add permeabilization lysis buffer to the treated cross-linked tissue sample, incubate fully until permeabilization is complete, and then add restriction endonuclease to perform in situ enzymatic digestion of DNA in the sample; (3) After the in situ enzyme digestion reaction is completed, add biotin-modified dNTPs to specifically label the ends of the enzyme digestion products; DNA ligase was then added to perform an ortho-ligation reaction to obtain the ligation product; Proteinase K was added to the ligation product, and after digestion, the cross-linking between DNA and protein was broken, and the purified DNA product was finally recovered. (4) The purified DNA product was cut using Tn5 transposase to obtain the target DNA fragment; Streptavidin-coated magnetic beads were used to specifically capture biotin-modified DNA fragments, followed by PCR amplification and fragment screening to obtain the target fragments and complete the construction of Hi-C libraries. (5) Select the capture probe and the Hi-C library for liquid-phase hybridization capture, and use streptavidin-coated magnetic beads to specifically adsorb the promoter-related long-range interaction fragments of the bound probe; The adsorption-obtained interaction fragments were amplified by PCR and purified to construct the Capture Hi-C library.

[0010] Preferably, the decrosslinking agent is a glycine solution; The permeabilization lysis buffer is sodium dodecyl sulfate; The restriction endonuclease is DpnⅡ.

[0011] A detection kit for targeting remote interaction sites of the pig whole genome promoter includes the aforementioned targeting capture probe, and one or more of the following: permeabilization lysis buffer, proteinase K, washing buffer, rinsing buffer, and elution buffer.

[0012] Application of a detection kit targeting remote interaction sites of the porcine whole-genome promoter in the construction and detection of high-throughput targeted capture libraries of remote interaction sites of the porcine whole-genome promoter.

[0013] Compared with the prior art, the present invention has at least the following technical effects: This invention provides a capture probe, kit, and applications for targeting remote interaction sites of the pig genome promoter. This technology belongs to the interdisciplinary field of biotechnology and three-dimensional genomics. Its successful development not only fills the technological gap in pig-specific Capture Hi-C probes, providing an efficient, precise, and economical technical means for three-dimensional interaction research of the pig genome, but also provides important support for functional analysis of the pig genome, the discovery of genetic mechanisms of complex traits, and innovation in molecular breeding technology. It has significant academic value and broad application prospects.

[0014] This capture probe, targeting remote interaction sites of promoters across the entire pig genome, can efficiently enrich and accurately detect remote interaction fragments between promoters and regulatory elements in the pig genome. This improves the feasibility and reliability of research on remote interactions in the pig genome and provides important technical support for pig genome functional studies, the analysis of genetic mechanisms of complex traits, and innovation in molecular breeding technologies.

[0015] This technology is a remote interaction target sequence capture system based on the hybridization principle. The kit uses capture probes specifically designed for the promoter region of the entire porcine genome. After hybridization with conventional Hi-C library sequences, it can efficiently enrich a large number of regulatory element sequences that interact with the promoter region. Subsequent data analysis can accurately capture the remote interaction loop structure between the promoter region and the regulatory element.

[0016] The kit of this invention optimizes probe targeting for porcine genome features, enabling precise capture of promoter-remote interaction sequences. Furthermore, with increasing sequencing depth, it effectively amplifies previously weak interaction signals, significantly improving the detection efficiency of low-abundance interaction information. This provides more comprehensive and in-depth technical support for the study of interaction mechanisms in promoter regions, and is suitable for scenarios such as porcine genome functional analysis and exploration of molecular mechanisms related to genetic breeding.

[0017] This technology also includes the following advantages: 1. First-ever design of specific capture probes targeting the promoter region of the entire porcine genome: Combining porcine genome characteristics, highly specific probes were designed with proximity to restriction endonuclease cleavage sites and GC content controlled at 20%-60%. A single promoter dual-probe design strategy was adopted, which solved the technical bottleneck of the lack of porcine-specific Capture Hi-C probes in the existing technology, filled the gap in three-dimensional interaction targeting detection tools for the entire porcine genome promoter region, and provided core tool support for porcine three-dimensional genome research.

[0018] 2. Efficient enrichment of long-range interaction fragments between promoters and regulatory elements: By specifically enriching long-range interaction fragments between promoters and regulatory elements across the entire porcine genome using probes, non-specific binding interference is effectively avoided, significantly improving the resolution and accuracy of interaction detection. Compared to traditional techniques such as Hi-C, this method can more accurately capture low-abundance, highly specific long-range interaction signals.

[0019] 3. Targeted strategies significantly improve detection efficiency and economic benefits: By utilizing targeted enrichment strategies, redundant data and excessive costs associated with indiscriminate whole-genome sequencing are avoided, significantly reducing experimental costs while maintaining high-resolution interaction detection. This method provides an economical, efficient, and scalable technical means for pig genetic breeding and functional genomics research, powerfully promoting the research and application of pig functional genomics. Attached Figure Description

[0020] Figure 1 A schematic diagram illustrating the design of a capture probe for the promoter region of the entire pig genome and the principle of Capture Hi-C. Figure 2 Here is a flowchart for Capture Hi-C's offline data analysis. Figure 3Capture a genome-wide Hi-C interaction map; Figure 4 A detailed schematic diagram illustrating the implementation of the case identification loop; Figure 5 This is a schematic diagram illustrating the analysis of loop length distribution characteristics; Figure 6 A schematic diagram illustrating the statistical distribution of the number of interactions associated with a single probe; Figure 7 This is a statistical diagram illustrating the percentage of probe interaction types. Detailed Implementation

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the present invention will be briefly introduced below in conjunction with the accompanying drawings and descriptions of the embodiments or the prior art. Obviously, the following description of the structure of the accompanying drawings is only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. It should be noted that the description of these embodiments is for the purpose of helping to understand the present invention, but does not constitute a limitation of the present invention.

[0022] See the flowchart of the implementation method of this invention. Figure 1 .like Figure 1 The diagram shown illustrates the design of a pig whole-genome promoter region capture probe and the Capture Hi-C principle.

[0023] In this process, the sample is first lysed and cross-linked with formaldehyde. DnpII restriction endonuclease fragments the genomic DNA and completes in situ neighbor-to-neighbor ligation. Then, the sample is constructed using conventional Hi-C library, and DNA fragments that interact remotely with the promoter region are enriched by specific probes. These fragments are then purified and enriched again using streptavidin magnetic beads, ultimately resulting in the construction of a high-quality promoter-targeted Capture Hi-C library.

[0024] One specific embodiment of the present invention is as follows: The application of a capture probe targeting long-range interaction sites of the porcine whole-genome promoter in the construction of high-throughput targeted capture libraries includes the following steps: (1) After thoroughly grinding fresh or liquid nitrogen-preserved porcine adipose tissue into a fine powder in liquid nitrogen, accurately weigh 2g of tissue sample, add DPBS solution containing 1% formaldehyde solution, and crosslink at room temperature for 15 minutes. Then add glycine solution with a final concentration of 0.125M, gently rotate at room temperature for 5 minutes to terminate the crosslinking reaction, centrifuge and discard the supernatant, retaining the precipitate; (2) Add cell lysis buffer containing 0.3%-0.5% sodium dodecyl sulfate to the precipitate from step (1), and incubate on ice for 10 minutes to permeate the cell membrane and nuclear membrane. Then terminate the lysis with a final concentration of 2% Triton X-100, and add an appropriate amount of Dpn II restriction endonuclease for digestion at 37 degrees Celsius. (3) Centrifuge the enzyme digestion product from step (2), discard the supernatant, add biotin-modified dNTPs, and perform end repair and biotin labeling; then add DNA ligase to connect the ends of spatially adjacent DNA fragments. (4) Add proteinase K to the ligation product of step (3) and incubate overnight at 65°C to reverse cross-linking and digest the protein, while purifying and extracting the DNA sample.

[0025] (5) The DNA from step (4) is fragmented using Tn5 transposase and an adapter sequence is added. The fragmented DNA is then purified and recovered. (6) Use streptavidin-coated magnetic beads to capture the biotin-modified fragment in step (5) and construct a Hi-C library by PCR amplification and purification; (7) Block the adapter sequences at both ends of the Hi-C library in step (6), and use the designed probe targeting the promoter region of the whole pig genome to fully hybridize and capture the probe at 65°C so that the probe can effectively bind to the target promoter sequence; (8) After hybridization, streptavidin magnetic beads are used to capture the DNA complex bound to the biotinylated probe in step (7) and to specifically adsorb fragments that have long-range interactions with the promoter region. (9) The specific sequence captured by the probe in step (8) is amplified and purified by PCR to enrich the target fragment and finally complete the preparation of the entire Capture Hi-C library. (10) Library quality control, sequencing, and data analysis.

[0026] (11) After the data is downloaded, analyze it according to the Capture Hi-C analysis process (e.g. Figure 2 As shown in Table 1, data processing was performed using the HiCUP analysis workflow, and the interaction strength was calculated using the CHiCAGO analysis workflow. Basic quality control statistics were performed on the Capture Hi-C sequencing data, and the results are shown in Table 1.

[0027] Table 1. Example of data quality control and statistical results description

[0028] The results are shown in Table 1. After filtering, alignment, effective pair screening, and redundancy removal, 96,455,765 and 92,538,883 duplicate effective interaction pairs were obtained in the two examples, respectively, indicating sufficient data volume and acceptable quality. According to interaction distance and type, interactions with cis>10kbp accounted for the highest proportion (approximately 58.7%), interactions with cis<10kbp accounted for approximately 7.7%-7.8%, and trans interactions accounted for approximately 33.5%-33.6%, showing good consistency among samples. The Capture Hi-C library constructed by the method of this invention is stable in quality and has good reproducibility, and can be used for subsequent genomic three-dimensional structure analysis and target gene regulatory relationship studies.

[0029] like Figure 3 The image shows a genome-wide Capture Hi-C interaction map; like Figure 4 The diagram shown is a detailed representation of the identification loop.

[0030] The results are as follows Figure 3 As shown, the analyzed data are visualized. Both examples present typical chromatin spatial interaction patterns, with interaction signals evenly distributed along the genome region. The morphology and density highly overlap, verifying the stability and reproducibility of this method.

[0031] The results are as follows Figure 4 As shown, further focusing on local interaction features, in a specific region of chromosome 3 (chr3:97,931,699) In the high-resolution interaction maps of 98,518,101, the interaction peak positions and signal intensities of the two sets of examples are highly conserved and significantly enriched near functional gene sites, suggesting a spatial coupling relationship between local three-dimensional conformation and gene regulation. At the same time, the two sets of samples show subtle differences in interaction intensity and clustering degree, indicating that this method can accurately capture and analyze sample-specific chromatin three-dimensional conformation and potential regulatory mechanisms.

[0032] like Figure 5 The diagram shown is a schematic diagram of the analysis of loop length distribution characteristics; like Figure 6 The figure shows a schematic diagram of the statistical distribution of the number of interactions associated with a single probe.

[0033] The results are as follows Figure 5 As shown, analysis of the loop length distribution characteristics reveals a typical short-range interactive enrichment trend. The number of loops decreases significantly with increasing physical distance, with loops in the 0–250 kb range being absolutely dominant, and very few long-distance loops. This distribution is consistent with typical biological characteristics of a three-dimensional genome.

[0034] The results are as follows Figure 6The results show that most probes participate in only limited chromatin interactions, while a few probes can act as core nodes to participate in a large number of interactions, forming a distribution pattern of "a few high-frequency interaction probes and a majority of low-frequency interaction probes", suggesting that these high-frequency probes may play a key hub role in the three-dimensional genome regulatory network.

[0035] like Figure 7 The figure shown is a statistical diagram illustrating the percentage of probe interaction types.

[0036] Subsequently, statistics on the proportion of probe interaction types were compiled, and the results were combined with... Figure 7 The results show that: cis-target region interaction accounted for the highest proportion, reaching 98.65%, with cis-regulation being dominant, reflecting the specificity and accuracy of the experimental system.

[0037] These results indicate that the chromatin interactions captured by this method are highly enriched in the probe-target region of cis-acting interactions, which is consistent with the characteristics of cis-regulation dominance in the three-dimensional genome, and also demonstrates the specificity and accuracy of the experimental system.

[0038] This invention designs probe sequences targeting the GATC restriction site of the entire pig genome. The GC content of the probe sequences is in the range of 20-60%. Two probes are designed for the promoter region of the entire pig genome, each probe is 120 bp in length, for a total of 47,027 probe sequences.

[0039] The process of designing and preparing probes in this invention is as follows: (1) First, the region from 1500bp upstream to 500bp downstream (total 2000bp) of the longest transcript TSS of each gene in the pig genome is defined as the promoter of that gene; (2) Restriction fragments corresponding to the restriction sites (GATC) are screened from each promoter, and fragments with a length >120bp are retained; a probe sequence is designed at both ends of the qualified restriction fragments. These sequences will hybridize and complement the target sequence in subsequent experiments due to the principle of complementary base pairing. (3) Each probe sequence must meet the following requirements: length 120bp, cumulative repeat sequence length less than 30bp, GC content between 20% and 60%, and good specificity. Probes that do not meet the requirements will be filtered out, and finally two high-quality probe sequences are retained for each promoter. (4) After summarizing all the probe sequences, we entrust a commercial company to use a high-throughput DNA synthesis platform to synthesize the sequences. The synthesized probes can be used for the capture experiment of Hi-C library.

[0040] This invention designs sequences for targeting and capturing remote interaction sites of the pig whole genome promoter. The inventors specifically explored a total of 47,027 sequences in experiments. The first 30 sequences of each of chromosomes 1-10 were extracted, as well as the first 15 sequences of each of chromosomes 11-18, X, and Y, totaling 600 nucleotide sequences.

[0041] The sequence list is as follows:

[0042] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A capture probe targeting long-range interaction sites of the porcine whole-genome promoter, characterized in that, The capture probes are two specific probes designed for each promoter region of the whole pig genome, and the nucleotide sequences of the probes are shown in SEQ ID NO.1-600; The GC content of the capture probe sequence is 20-60%; Each of the aforementioned capture probes is 120 bp in length.

2. The capture probe according to claim 1, characterized in that, The capture probe is located near the GATC restriction site within the promoter region of the pig genome.

3. The capture probe according to claim 1, characterized in that, The capture probe also includes a modification group; The modifying group is located at the 5' or 3' end of the probe and includes one or more of biotin, fluorescein, carboxyl modifying group, thiol modifying group and phosphorylation modifying group.

4. An application of the capture probe as described in any one of claims 1 to 2 in the construction of a high-throughput targeted capture library, characterized in that, Includes the following steps: (1) Select fresh or liquid nitrogen-preserved pig tissues and grind them into powder in liquid nitrogen. Then, use a formaldehyde solution with a mass concentration of 1-2% to cross-link the tissue samples. After the cross-linking is completed, add a de-cross-linking agent to terminate the cross-linking and collect the cross-linked tissue samples after treatment. (2) Add permeabilization lysis buffer to the treated cross-linked tissue sample, incubate fully until permeabilization is complete, and then add restriction endonuclease to perform in situ enzymatic digestion of DNA in the sample; (3) After the in situ enzyme digestion reaction is completed, add biotin-modified dNTPs to specifically label the ends of the enzyme digestion products; DNA ligase was then added to perform an ortho-ligation reaction to obtain the ligation product; Proteinase K was added to the ligation product, and after digestion, the cross-linking between DNA and protein was broken, and the purified DNA product was finally recovered. (4) The purified DNA product was cut using Tn5 transposase to obtain the target DNA fragment; Streptavidin-coated magnetic beads were used to specifically capture biotin-modified DNA fragments, followed by PCR amplification and fragment screening to obtain the target fragments and complete the construction of Hi-C libraries. (5) Select the capture probe and the Hi-C library for liquid-phase hybridization capture, and use streptavidin-coated magnetic beads to specifically adsorb the promoter-related long-range interaction fragments of the bound probe; The adsorption-obtained interaction fragments were amplified by PCR and purified to construct the Capture Hi-C library.

5. The application according to claim 4, characterized in that, The decrosslinking agent is a glycine solution; The permeabilization lysis buffer is sodium dodecyl sulfate; The restriction endonuclease is DpnⅡ.

6. A detection kit targeting long-range interaction sites of the porcine whole-genome promoter, characterized in that, It includes the targeted capture probe as described in any one of claims 1 to 3, and one or more of the following: permeabilization lysis buffer, proteinase K, washing buffer, rinsing buffer, and elution buffer.

7. The application of the detection kit as described in claim 6 in the construction and detection of a high-throughput targeted capture library of remote interaction sites of the porcine whole-genome promoter.

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