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CN-122012494-A - Alu element hypomethylation region capture probe and application thereof

CN122012494ACN 122012494 ACN122012494 ACN 122012494ACN-122012494-A

Abstract

The invention relates to an Alu element hypomethylation region capture probe and application thereof. The invention utilizes Alu hypomethylation capture probes to carry out capturing of hypomethylation regions of three families of genes of Alu elements in a whole genome range through a non-sulfite transformation strategy. After high-throughput sequencing, an Alu hypomethylation map can be established, and a new way is provided for disease screening by utilizing Alu repeat elements.

Inventors

  • WANG MEICONG
  • GONG YANAN
  • ZHANG XIAOXIAO
  • WAN JIAWEI
  • CHAI JUN
  • LI WEIZHENG
  • ZHOU XUJIAN
  • WANG DONGXU
  • XIE WEIJUAN
  • HU YAO

Assignees

  • 浙江默乐生物科技有限公司
  • 江苏默乐生物科技股份有限公司

Dates

Publication Date
20260512
Application Date
20241111

Claims (8)

  1. 1. A probe combination based on a methylation CpG binding protein affinity capture technology and specifically recognizing Alu element hypomethylation regions is characterized in that the nucleotide sequences of the probe combination are respectively shown as SEQ ID NO. 1-SEQ ID NO. 9.
  2. 2. A kit for specifically identifying Alu element hypomethylation regions based on methylation CpG binding protein affinity capture technology is characterized by comprising probes respectively having nucleotide sequences shown in SEQ ID NO. 1-SEQ ID NO. 9.
  3. 3. The kit of claim 2, further comprising a methylated CpG binding protein selected from one or more of MeCP2, MBD1, MBD2, MBD3, MBD 4.
  4. 4. A method of capturing an Alu element hypomethylated region comprising the steps of: s1, after the methylation CpG binding protein is combined with magnetic beads, carrying out antigen-antibody reaction with a connection product obtained after the treatment of a DNA library construction method, and collecting hypomethylation DNA fragments; s2, carrying out PCR amplification on the hypomethylated fragments, and purifying to obtain a hypomethylation enrichment library; S3, performing liquid phase hybridization capture reaction on probes respectively having nucleotide sequences shown in SEQ ID NO. 1-SEQ ID NO. 9 and the hypomethylation enrichment library to obtain the objective Alu element hypomethylation fragment.
  5. 5. The capture method of claim 4, wherein in step S1, the methylated CpG binding protein is selected from one or more of MeCP2, MBD1, MBD2, MBD3, MBD 4.
  6. 6. The method according to claim 4, wherein in step S3, the liquid phase hybridization is performed at a reaction temperature of 65℃for 4 to 16 hours, and no human placenta DNA is added during the liquid phase hybridization.
  7. 7. Use of a probe combination according to claim 1 or a kit according to claim 2 or a capture method according to claim 4 for screening markers for disease prediction, diagnosis or prognosis evaluation.
  8. 8. The use according to claim 7, wherein the disease is colorectal cancer.

Description

Alu element hypomethylation region capture probe and application thereof Technical Field The invention relates to the technical field of gene sequencing, in particular to an Alu element hypomethylation region targeted capture probe, a kit, a capture method and application thereof. Background DNA methylation generally refers to the methylation state of the cytosine carbon number 5 in a genomic CpG. As an important epigenetic regulatory mechanism, it plays an important role in development and disease progression. DNA methylation abnormalities are an important early event in the development and progression of cancer. In normal human DNA, 2% -7% of the cytosines are methylated, with CpG being the most important methylation site, which presents an uneven distribution in the genome, with hypermethylated, hypomethylated and unmethylated regions. The sequence density in certain regions of the genome, such as the promoter region, 5' UTR (untranslated region) and the first exon CpG of the gene, is very high. Typically, promoter DNA hypermethylation is associated with gene silencing, while hypomethylation is associated with gene activation. Numerous studies have shown that the inactivation of an oncogene is directly linked to hypermethylation of CpG islands in the promoter region of the gene, resulting in the occurrence of cancer. Thus, the current epigenetic studies and clinical applications are focused on hypermethylation studies, which tend to ignore the study of repetitive elements. The complexity of repetitive element studies far exceeds that of functional genes, and current high resolution DNA methylation profiles contain few repetitive elements, and the design of targeted capture probes by whole gene methylation sequencing and methylation is avoided. The Alu repeat sequence in human is a member of SINE family, and is a series of related sequences distributed in a scattered manner, each about 280 bp. Each member has cleavage sites for Alu restriction enzymes at both ends and is thus named. Alu elements have 3 families, 9 subfamilies, respectively the J family (Jo and Jb subfamilies), the S family (Sg, sc, sx, sp and Sq subfamilies), and the Y family (Yb 8 and Ya5, yab subfamilies). Alu elements are distributed throughout the human chromosome and specific Alu sequences exist in the promoter, 5' -UTR, first exon and intron (introns) of 10-15% of genes. Alu elements are widely distributed at CpG sites, with methylation occurring across the genome, accounting for nearly one quarter of Alu methylation. For carrying out Alu methylation analysis in the whole genome range, the total level change of Alu methylation is quantitatively evaluated by mainly adopting a sulfite treatment mode at present through Alu-PCR, and no report is yet made on specific Alu locus analysis. Conventional Alu-PCR techniques naturally lose much information when analyzed for free fragments, especially cfDNA. The cfDNA fragment distribution peak is generally about 166-167bp, the Alu repeated sequence is generally about 280bp, and only a part of information can be obtained by a PCR method. In methylation analysis in the whole genome, a technique for capturing Alu is more required. The conventional probe hybridization capture technology flow is not suitable for methylation analysis of Alu because the repetitive sequence analysis in the gene is automatically shielded due to the addition of the human cot DNA. In addition, the main methylation analysis technique is mainly performed by converting unmethylated cytosine into uracil by bisulfite treatment, while methylated cytosine remains unchanged in base. The manner of bisulfite treatment is very costly in terms of degradation loss of DNA, especially for low initial sample types such as cfDNA, so that much methylation information cannot be detected by subsequent procedures, while clinically blood collection is often limiting and the difficulty of low initial amounts cannot be addressed directly by large amounts of blood collection. Meanwhile, the bisulfite treatment greatly changes the base distribution, so that the problem of bias in sequencing is remarkable, and sequencing correction is required for an unbalanced library. Disclosure of Invention The invention aims to provide an important supplementary means for early cancer screening or medical intervention means evaluation and prognosis detection by obtaining the distribution of Alu element hypomethylation. In order to accomplish the object, the present invention provides the following technical solutions: The first aspect of the invention provides a probe combination, and the nucleotide sequences of the probe combination are respectively shown as SEQ ID NO. 1-SEQ ID NO. 9. The probe combination is based on methylation CpG binding protein affinity capture technology, and specifically recognizes Alu element hypomethylation regions. The probe combination is a liquid phase hybridization capture probe based on a non-sulfite conversion strategy. Alu cons