Alu element low-methylation region capture probe and application thereof

Abstract

The invention relates to an Alu element low-methylation region capture probe and application thereof. According to the invention, through a non-sulfite conversion strategy, an Alu low-methylation capture probe is utilized to carry out low-methylation region capture of three family genes of an Alu element in a whole genome range. After high-throughput sequencing, an Alu low-methylation map can be established, and a new way is provided for disease screening by using Alu repetitive elements.

Description

Technical Field

[0001] This invention relates to the field of gene sequencing technology, specifically to a probe for targeted capture of hypomethylated regions of Alu elements, a kit, a capture method, and their applications. Background Technology

[0002] DNA methylation generally refers to the methylation state of the 5th carbon of cytosine in the CpG of the genome. As an important epigenetic regulatory mechanism, it plays a crucial role in development and disease progression. Abnormal DNA methylation is an important early event in the occurrence and development of cancer.

[0003] In normal human DNA, 2%–7% of cytosine is methylated, with CpG being the most important methylation site. It exhibits uneven distribution across the genome, including hypermethylated, hypomethylated, and unmethylated regions. In certain regions of the genome, such as the promoter region, 5'UTR (untranslated region), and the first exon, the CpG sequence density is very high. Generally, hypermethylation of promoter region DNA is associated with gene silencing, while hypomethylation is associated with gene activation. Numerous studies have shown a direct link between the inactivation of tumor suppressor genes and the hypermethylation of CpG islands in the promoter region of these genes, leading to cancer development.

[0004] Therefore, current epigenetic research and clinical applications focus on the study of hypermethylation, often neglecting the study of repetitive elements. The complexity of studying repetitive elements far exceeds that of functional genes. Current high-resolution DNA methylation maps almost never include repetitive elements. By using whole-genome methylation sequencing and designing methylation-targeting capture probes, the design of repetitive elements can be avoided.

[0005] Alu repeat sequences in humans are members of the SINE family and are a series of dispersed, related sequences, each approximately 280 bp in length. Each member has cleavage sites for Alu restriction enzymes at both ends, hence its name. There are three families and nine subfamilies of Alu elements: the J family (Jo and Jb subfamilies), the S family (Sg, Sc, Sx, Sp, and Sq subfamilies), and the Y family (Yb8, Ya5, and Yab subfamilies). Alu elements are distributed throughout all human chromosomes, and specific Alu sequences are found in the promoter, 5'-UTR, first exon, and intron of 10–15% of genes. Alu elements are widely distributed at CpG sites, and Alu methylation accounts for nearly a quarter of all genome-wide methylation.

[0006] Currently, genome-wide Alu methylation analysis primarily utilizes sulfite treatment followed by Alu-PCR to quantitatively evaluate the overall Alu methylation level. However, analysis of specific Alu sites remains unreported. Conventional Alu-PCR naturally loses significant information when analyzing cell-free fragments, especially cfDNA. This is because cfDNA fragment peaks are typically around 166–167 bp, while Alu repetitive sequences are generally around 280 bp, meaning PCR can only obtain partial information. For genome-wide methylation analysis, more comprehensive techniques capable of capturing Alu methylation are needed. Conventional probe hybridization capture techniques, due to the inclusion of human cot DNA, automatically mask repetitive sequence analysis, making them unsuitable for Alu methylation analysis. Furthermore, mainstream methylation analysis techniques mainly rely on bisulfite treatment to convert unmethylated cytosine to uracil, while leaving methylated cytosine unchanged. Bisulfite treatment causes significant DNA degradation, especially in low-starting-volume samples such as cfDNA. Excessive degradation prevents many methylation markers from being detected in subsequent procedures. However, clinical blood collection is often limited, making it impossible to directly address the low-starting-volume problem by drawing large amounts of blood. Furthermore, bisulfite treatment drastically alters the base distribution, exacerbating sequencing bias and necessitating sequencing correction for imbalanced libraries. Summary of the Invention

[0007] The purpose of this invention is to provide an important supplementary method for early cancer screening, evaluation of medical interventions, and prognostic detection by obtaining the distribution of hypomethylation of Alu elements. To achieve this purpose, this invention provides the following technical solution:

[0008] The first aspect of this invention provides a probe assembly whose nucleotide sequences are shown in SEQ ID NO:1 to SEQ ID NO:9. This probe assembly is based on methylated CpG binding protein affinity capture technology and specifically recognizes the hypomethylated region of the Alu element.

[0009] This probe ensemble is a liquid-phase hybridization capture probe based on a non-sulfite conversion strategy. Conserved Alu sequences and specific sequences from three families (J, S, and Y families) were included in the design.

[0010] A second aspect of the present invention provides a kit for specifically recognizing hypomethylated regions of Alu elements based on methylated CpG binding protein affinity capture technology, comprising probes having nucleotide sequences shown in SEQ ID NO:1 to SEQ ID NO:9 respectively.

[0011] In some embodiments of the present invention, the kit further includes a methylated CpG binding protein selected from one or more of MeCP2, MBD1, MBD2, MBD3, and MBD4.

[0012] A third aspect of the present invention provides a method for capturing hypomethylated regions of Alu elements, comprising the following steps:

[0013] S1 employs methylated CpG-binding protein affinity capture technology, where methylated CpG-binding proteins are bound to magnetic beads and then reacted with the ligation products obtained after DNA library construction to collect hypomethylated fragments.

[0014] S2, the hypomethylated fragments were amplified by PCR and purified to obtain a hypomethylated enriched library.

[0015] S3, using probes with nucleotide sequences shown in SEQ ID NO:1 to SEQ ID NO:9 respectively, a liquid-phase hybridization capture reaction is performed with a hypomethylation enrichment library to obtain the hypomethylation fragment of the target Alu element.

[0016] In some embodiments of the present invention, the above-described capture method further includes a pretreatment step: S0, extracting DNA, constructing a DNA library, and obtaining a ligation product.

[0017] In some embodiments of the present invention, the above-described capture method further includes a post-processing step: S4, purifying the target Alu element hypomethylated fragment and then performing PCR amplification, followed by further purification to obtain an amplified Alu element hypomethylated library, and then performing high-throughput sequencing. Preferably, the high-throughput sequencing uses Illumina next-generation sequencing.

[0018] In some embodiments of the present invention, in step S1, the methylated CpG binding protein is selected from one or more of MeCP2, MBD1, MBD2, MBD3, and MBD4.

[0019] In some embodiments of the present invention, in step S3, the liquid-phase hybridization capture reaction temperature is 65°C; the reaction time is 4 to 16 hours; and no Human Cot DNA is added during the liquid-phase hybridization capture reaction.

[0020] The fourth aspect of the present invention provides the application of the probe combination of the first aspect, the kit of the second aspect, or the method for capturing the hypomethylated region of the Alu element of the third aspect in screening biomarkers for disease prediction, diagnosis, or prognostic assessment.

[0021] Preferably, the disease includes, but is not limited to, cancer and malignant tumors, such as lung cancer, colorectal cancer, stomach cancer, breast cancer, and colorectal cancer. More preferably, the disease is colorectal cancer.

[0022] In some embodiments of the present invention, when screening biomarkers for disease prediction, diagnosis or prognostic assessment using the method for capturing the hypomethylated region of Alu element in the third aspect of the present invention, the method further includes the step of obtaining a hypomethylated region map of Alu element obtained by high-throughput sequencing of the hypomethylated fragment of Alu element obtained by the method for capturing the hypomethylated region of Alu element.

[0023] Beneficial effects of the present invention

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] Using this invention, the hypomethylation trend of Alu elements can be obtained, which can serve as an important supplementary means for early cancer screening, evaluation of medical interventions, and prognosis detection.

[0026] Using this invention, a hypomethylation spectrum of Alu can be obtained, and then specific Alu sequences that are significant for grouping early tumor occurrence or prognosis can be screened out. These sequences can be further designed into probes that can accurately and sensitively achieve early screening, early diagnosis, or recurrence detection of cancer.

[0027] Hypomethylation of Alu elements is likely one of the important biological mechanisms underlying methylation of promoters of nearby functional genes. This invention provides significant support for elucidating the molecular mechanisms by which functional genes play a role in tumorigenesis and development.

[0028] Specifically:

[0029] (1) This invention can achieve regional localization of the Alu whole genome hypomethylation map and obtain high-resolution information on hypomethylation regions;

[0030] (2) This invention avoids the problems of excessive degradation of bisulfite and sequencing bias. By enriching the methylated CpG binding protein MBD, it reverses the collection of hypomethylated fragments and obtains sufficient demethylated / hypomethylated region information, which is suitable for grouping and comparing different types of tumor samples.

[0031] (3) This invention is not only applicable to cfDNA samples, but also to tissue samples, including paraffin section samples and gDNA analysis in body fluid samples, and has a wide range of applications.

[0032] (4) This invention is based on probe hybridization capture, which has high throughput and can achieve simultaneous sequencing of multiple samples according to different adapters. Attached Figure Description

[0033] Figure 1 The characteristic sequence of Alu elements is shown.

[0034] Figure 2 The figure shows the percentage of the Alu region captured by the probe of this invention relative to all enriched peaks.

[0035] Figure 3 A partial view of the IGV plot showing the difference in Alu in cfDNA samples between colorectal cancer patients and normal healthy individuals using the probe of the present invention is shown (chr16:1290582-1290982).

[0036] Figure 4 A partial display of IGV plots showing the difference in Alu between colorectal cancer tissue and adjacent normal tissue samples detected using the probe of the present invention is shown (chr21:6438444-6438845). Detailed Implementation

[0037] Unless otherwise stated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by one of ordinary skill in the art or the field of application of such terms. While any sequences, substances, or materials similar to or equivalent to those disclosed herein may be used in practice, preferred sequences, substances, or materials are described herein.

[0038] In this invention, hypomethylation refers to the presence of fewer CpG methylation sites in DNA. DNA hypomethylation primarily affects repetitive elements; tumor cells typically exhibit both localized hypermethylation and widespread hypomethylation.

[0039] To make the technical problems solved, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to embodiments. The following examples are used to illustrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the following examples represent technologies discovered by the inventors that can be used to implement this invention, and therefore can be considered as preferred solutions for implementing this invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still obtaining the same or similar results, without departing from the spirit or scope of the invention.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent suppliers.

[0041] Example 1: Human Alu Element Hypomethylation Targeted Capture Probe

[0042] 1. Determine the conserved sequences of Alu elements and the differential sequences corresponding to the three families.

[0043] The inventors obtained the characteristic sequences of Alu elements by searching and analyzing existing reports on Alu elements, such as... Figure 1 As shown.

[0044] 2. Probe Design

[0045] Liquid-phase hybridization capture probe sequences based on a non-sulfite conversion strategy were designed using the website http: / / nadprobe.njnad.com / login.html. Conserved Alu sequences and specific sequences from three families (J, S, and Y families) were included in the design scope, resulting in nine probes. Their nucleotide sequences are shown in Table 1.

[0046] Table 1

[0047]

[0048]

[0049] 3. Synthesis of probes targeting the hypomethylation region of the Alu element

[0050] The probe was synthesized by Naonda (Nanjing) Biotechnology Co., Ltd.

[0051] The probes described above can be used to prepare kits for capturing hypomethylated regions of Alu elements.

[0052] Example 2: Application of probes targeting the hypomethylated region of the Alu element in early colorectal cancer screening markers.

[0053] This embodiment uses blood samples, cancerous tissue, and adjacent normal tissue samples from 160 clinical colorectal cancer patients from the First Affiliated Hospital of Zhejiang University, as well as blood samples from 160 patients with normal intestinal health and benign diseases.

[0054] 1. DNA extraction

[0055] (1) cfDNA extraction: 1-2 mL of isolated plasma sample was extracted with VAHTS Free-Circulating DNA MaxiKit (Cat.No.N903-03, Vazyme) to obtain cfDNA.

[0056] (2) gDNA extraction from leukocytes, cancer tissues, paraffin sections or body fluids can all be performed using commercially available kits.

[0057] 2. DNA fragmentation

[0058] DNA fragmentation can be performed using enzyme digestion methods, and commercially available genomic enzyme digestion kits can be used for fragmentation.

[0059] 3. Library Construction

[0060] Any commercially available library construction kit can be used. In this example, the VAHTS Universal Pro DNALibrary Prep Kit for illumina (Cat.No.ND608-02, Vazyme) was selected, and the cfDNA obtained in the aforementioned nucleic acid extraction steps was subjected to end repair, A-tailing, and ligation with adapters according to the instructions to obtain the ligation product; preferably, adapters with a unique UMI molecular identifier were used for the ligation reaction.

[0061] 4. MBD enrichment

[0062] Using the EpiXplore™ Methylated DNA Enrichment Kit (Cat. No. 631963, Takara), follow the instructions. After binding methylated CpG-binding proteins (e.g., MBD2 protein) to magnetic beads, perform an antigen-antibody reaction with the ligation product. The methylated DNA region binds to the antibody and magnetic beads; collect the supernatant in reverse order. Then, purify the unmethylated DNA region in the supernatant using magnetic beads, achieving reverse collection and enrichment of hypomethylated DNA fragments.

[0063] 5. Short-cycle PCR amplification and purification before capture

[0064] PCR short-cycle amplification was performed using KAPA HiFi Hot start Ready mix and Library Amplification Primer Mix according to the instructions, followed by purification to obtain a hypomethylated library.

[0065] 6. Liquid-phase hybridization capture

[0066] Liquid-phase hybridization capture was performed using NadPrep hybridization capture reagent (catalog number: REF1005101, NadPrep (Nanjing) Biotechnology Co., Ltd.). The total amount of hypomethylated library added for each hybridization capture reaction should be between 300 ng and 2 μg. Take all the hypomethylated library, not exceeding 2 μg, and add NadPrep reagent. Nano Blockers were dried in a vacuum concentrator preheated to 42°C at a speed of 1000 rpm. After drying, the prepared hybridization reaction solution and the self-designed Alu-panel (the Alu element hypomethylated targeted capture probe synthesized in Example 1) were added. The mixture was oscillated and centrifuged briefly. The hybridization was carried out at 95°C for 30 seconds and 65°C for Hold (100°C hot cover) for 4-16 hours. Then, the washed streptavidin magnetic beads were added to the hybridization system and incubated for 40 minutes, vortexing every 10 minutes to ensure complete resuspension of the magnetic beads.

[0067] It is particularly important to note that Human Cot DNA must not be added, otherwise the liquid phase hybridization capture reaction will fail.

[0068] After the hybridization capture reaction is complete, wash the bound magnetic beads with the four washing solutions provided in the kit, discarding any residual solution at each step; finally, add 20 μl of nuclease-free water and gently vortex to mix.

[0069] 7. Short-cycle PCR amplification and purification after capture

[0070] Using KAPA HiFi Hot start Ready mix and Library Amplification Primer Mix, PCR amplification after hybridization capture was performed according to the instructions for 11-13 cycles, followed by purification to obtain the Alu hypomethylated library.

[0071] 8. Library pooling, preparation for computer lab session

[0072] Dilute the Alu capture library to 4 nM and mix it according to the required data volume, ensuring the total data volume does not exceed 120 G. The concentration of the library to be used in the system is 1.5 pM.

[0073] 9. Illumina NextSeq 550Dx sequencing

[0074] The sequencer used was an Illumina NextSeq 550Dx. Reagents used included High Output Reagent Cartridge v2 (REF:15057929, Illumina) (300 cycles), High Output Flow Cell Cartridge v2.5 (REF:20022408, Illumina), and Buffer Cartridge v2 (REF:15057941, Illumina). 1300 μl of the library was added to the sample space of the High Output Reagent Cartridge v2, and each reagent was added sequentially. Sequencing could then begin. This example used paired-end sequencing, with a total sequencing time of approximately 30 hours.

[0075] 10. Bioinformatics Analysis of Data After Deployment

[0076] Fastp (v0.22.0) was used for quality control of the data, retaining reads with Q20 > 90% and Q30 > 85%. The reads were aligned to the Hg38 reference genome using bwa, deduplicated, and then the DESeq and edgeR algorithms of the DiffBind tool were used to select significantly different markers. Finally, IGV was used for visualization analysis.

[0077] The probe designed in Example 1 has the following capture efficiency for Alu: Figure 2 As shown. For most samples, compared with the Hg38 reference genome, the capture efficiency of Alu elements is around 90%, indicating that the probe designed in this invention has extremely high capture efficiency.

[0078] IGV maps of regions showing differential enrichment of Alu hypomethylation in cfDNA samples from colorectal cancer and healthy individuals (chr16: 1290582-1290982) are shown below. Figure 3 Partially shown IGV plots illustrating the Alu difference between colorectal cancer tissue and adjacent normal tissue samples (chr21: 6438444-6438845). Figure 4 .

[0079] Depend on Figure 3 and Figure 4 It can be seen that there are significant differences in Alu hypomethylation profiles between colorectal cancer patients and non-colorectal cancer samples. Based on the analysis of Alu hypomethylation profiles in colorectal cancer patients and normal individuals, the genomic locations of some significantly different markers are shown in Table 2:

[0080] Table 2. Markers of significant differences between colorectal cancer patients and normal individuals.

[0081]

[0082]

[0083] Analyzing these differences in Alu elements in specific regions of the genome could provide new technological options for distinguishing between cancer patients and non-cancer patients.

[0084] The inventors further focused their analysis on Alu element markers near methylation genes associated with colorectal cancer, such as TSPAN3_AluSp, SEPTIN9_AluSx3, N4BP2_AluSz6, SDC2_AluYc, IRF4_AluSx, and TSPAN3_AluSq2, as shown in Table 3.

[0085] Table 3

[0086]

[0087]

[0088] In Table 3, TSPAN3_AluSp represents the AluSp subfamily near the TSPAN3 gene, SEPTIN9_AluSx3 represents the AluSx3 subfamily near the SEPTIN9 gene, N4BP2_AluSz6 represents the AluSz6 subfamily near the N4BP2 gene, SDC2_AluYc represents the AluYc subfamily near the SDC2 gene, IRF4_AluSx represents the AluSx subfamily near the IRF4 gene, and TSPAN3_AluSq2 represents the AluSq2 subfamily near the TSPAN3 gene.

[0089] In Table 3, the number 0 indicates that there are 0 Alu elements with different levels of hypomethylation, the number 1 indicates that there are 1 Alu elements with different levels of hypomethylation, and the number 2 indicates that there are 2 Alu elements with different levels of hypomethylation.

[0090] The cfDNA samples from colorectal cancer patients and normal individuals show significant differences. These findings provide strong candidate markers for the construction of early screening models and offer biological theoretical support for the methylation of functional methylated genes.

[0091] This document uses specific examples to illustrate the principles and implementation methods of the present invention. The above description of the implementation is only for the purpose of helping to understand the method and core ideas of the present invention. At the same time, for those skilled in the art, based on the ideas of the present invention, there will be changes in the specific implementation methods and application scope. Modifications and improvements to the present invention are possible without exceeding the concept and scope defined by the appended claims. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A probe array based on methylated CpG-binding protein affinity capture technology that specifically recognizes hypomethylated regions of Alu elements, characterized in that, The nucleotide sequences of the probe combinations are shown in SEQ ID NO:1 to SEQ ID NO:9, respectively.

2. A kit for specifically recognizing hypomethylated regions of Alu elements based on methylated CpG-binding protein affinity capture technology, characterized in that, Including probes having the nucleotide sequences shown in SEQ ID NO:1 to SEQ ID NO:9 respectively.

3. The kit according to claim 2, characterized in that, It also includes methylated CpG-binding proteins, which are selected from one or more of MeCP2, MBD1, MBD2, MBD3, and MBD4.

4. A method for capturing hypomethylated regions of Alu elements, comprising the following steps: S1, after binding methylated CpG-binding proteins to magnetic beads, they are subjected to antigen-antibody reaction with the ligation products obtained by DNA library construction methods to collect hypomethylated DNA fragments. S2, the hypomethylated fragments were amplified by PCR and purified to obtain a hypomethylated enriched library. S3, using probes with nucleotide sequences shown in SEQ ID NO:1 to SEQ ID NO:9 respectively, a liquid-phase hybridization capture reaction is performed with a hypomethylation enrichment library to obtain the hypomethylation fragment of the target Alu element.

5. The capture method as described in claim 4, characterized in that, In step S1, the methylated CpG binding protein is selected from one or more of MeCP2, MBD1, MBD2, MBD3, and MBD4.

6. The capture method as described in claim 4, characterized in that, In step S3, the liquid-phase hybridization capture reaction temperature is 65°C; the reaction time is 4–16 hours; and no human placental DNA is added during the liquid-phase hybridization capture reaction.

7. The use of the probe combination of claim 1, the kit of claim 2, or the capture method of claim 4 in screening biomarkers for disease prediction, diagnosis, or prognostic assessment.

8. The application as described in claim 7, characterized in that, The disease in question is colorectal cancer.

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