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CN-121975913-A - Construction method and application of nucleic acid sequence for cfDNA multiplex gene methylation detection

CN121975913ACN 121975913 ACN121975913 ACN 121975913ACN-121975913-A

Abstract

The invention discloses a construction method of a nucleic acid sequence for cfDNA multiplex gene methylation detection and application thereof. The construction method comprises the steps of carrying out enzyme digestion on cfDNA to be detected by using methylation sensitive restriction enzyme to obtain an enzyme digestion section, connecting a connector to the incision end of the enzyme digestion section by using ligase to form a connector product, and carrying out PCR (polymerase chain reaction) amplification by using the connector product as a template to construct a recombinant nucleic acid sequence for detecting a methylation site of a target gene. The construction method can be used for methylation qPCR analysis of cfDNA level and quantitative analysis of low-abundance specimens, and has the advantages of small sample demand, wide coverage range of methylation analysis and high methylation enrichment efficiency.

Inventors

  • LUO DIXIAN
  • LIU QUAN
  • LIU YARU
  • WANG JING
  • Wen Xiaosha
  • He Tuping

Assignees

  • 深圳市罗湖区人民医院

Dates

Publication Date
20260505
Application Date
20260109

Claims (10)

  1. 1. A construction method of a nucleic acid sequence for cfDNA multiplex gene methylation detection is characterized by comprising the steps of performing enzyme digestion on cfDNA to be detected by using methylation-sensitive restriction enzymes to obtain enzyme fragments, connecting a linker to the cut ends of the enzyme fragments by using a ligase to form a linker product, and performing PCR amplification by using the linker product as a template to construct a recombinant nucleic acid sequence for target gene methylation site detection.
  2. 2. The method of constructing a nucleic acid sequence for cfDNA multiplex gene methylation detection according to claim 1, wherein the methylation sensitive restriction enzyme comprises AciI and/or HhaI.
  3. 3. The method for constructing a nucleic acid sequence for cfDNA multiplex gene methylation detection according to claim 1 or 2, wherein the nucleotide sequence protruding from the 5' -end of the cohesive end of the adaptor is CG.
  4. 4. A method of constructing a nucleic acid sequence for cfDNA multiplex gene methylation detection according to any of claims 1 to 3, wherein said ligase comprises a T4 DNA ligase.
  5. 5. The method for constructing a nucleic acid sequence for cfDNA multiplex gene methylation detection according to any one of claims 1 to 4, wherein the source of cfDNA to be detected comprises a biological sample and/or an artificially synthesized sample, and the biological sample comprises any one of plasma, serum, tissue or cells.
  6. 6. Use of the method for constructing nucleic acid sequences for cfDNA multiplex gene methylation detection according to any one of claims 1-5 for preparing methylation detection products.
  7. 7. A method for methylation detection of cervical cancer-associated genes, the method comprising the steps of: (1) Processing a cfDNA sample to be tested by adopting the construction method of the nucleic acid sequence for cfDNA multiplex gene methylation detection according to any one of claims 1-5, so as to obtain a recombinant nucleic acid sequence; (2) Taking the recombinant nucleic acid sequence as a template, and carrying out quantitative multiplex methylation specific PCR amplification detection by using a primer and a fluorescent probe for detecting cervical cancer related genes; (3) And judging the methylation state of the gene based on the amplification detection result.
  8. 8. The method for methylation detection of cervical cancer-associated genes according to claim 7, wherein the cervical cancer-associated genes comprise any one or a combination of at least two of PAX1, SOX1, RXFP3.1, RXFP3.2 or ZNF 671.
  9. 9. The method for methylation detection of cervical cancer-associated genes according to claim 7 or 8, wherein the nucleic acid sequence of the primer comprises the sequences shown in SEQ ID NO.1-SEQ ID NO.10, and the nucleic acid sequence of the fluorescent probe comprises the sequences shown in SEQ ID NO.11-SEQ ID NO. 15; Preferably, the fluorescent probe comprises a fluorescent group at the 5 'end and a quenching group at the 3' end; Preferably, the fluorophore comprises any one or a combination of at least two of FAM, HEX, VIC or ROX; Preferably, the quenching group comprises any one or a combination of at least two of BHQ1, BHQ2 or MGB.
  10. 10. The method for methylation detection of cervical cancer-associated genes according to any one of claims 7 to 9, wherein the quantitative multiplex methylation-specific PCR amplification system comprises templates, primers, fluorescent probes and qPCR premix; Preferably, the concentration of the primer in the system is 5-10 mu M, and the concentration of the fluorescent probe is 5-10 mu M; preferably, the quantitative multiplex methylation specific PCR amplification procedure comprises (1) a pre-denaturation at 93-95℃of 30-40 s, (2) a denaturation at 93-95℃of 5-10 s, (2) an annealing at 55-60℃for 20-30 s, and an extension at 55-60℃for 20-30 s for a total of 40-45 cycles.

Description

Construction method and application of nucleic acid sequence for cfDNA multiplex gene methylation detection Technical Field The invention belongs to the technical field of biological medicine, and relates to a construction method and application of a nucleic acid sequence for cfDNA multiplex gene methylation detection. Background DNA methylation, one of the core mechanisms of epigenetic modification, plays a vital role in gene expression regulation, cell differentiation, and disease development and progression. In particular in the tumor field, hypermethylation of the promoter region of a particular gene is an important early molecular event. Therefore, the development of sensitive, accurate and efficient DNA methylation detection technology has great significance for early screening, diagnosis and prognosis monitoring of cancers. Circulating free DNA (cfDNA), especially circulating tumor DNA (ctDNA) derived from tumors, has become a very potential liquid biopsy target. However, cfDNA is very low in blood, is severely fragmented, and often contains large amounts of wild-type background DNA, which presents a significant challenge for detection of low abundance methylation signals therein. Currently, the mainstream detection technology of DNA methylation is mainly based on the following principles 1. Bisulfite treatment combined with sequencing/PCR method, which is the current gold standard. Which converts unmethylated cytosine (C) to uracil (U) by bisulfite, while methylated C remains unchanged, and is identified by subsequent sequencing or specific PCR. However, this method has non-negligible drawbacks including severe DNA damage, extremely high sample loss rate (typically > 90%), difficult to reach 100% conversion efficiency, and reduced sequence complexity. 2. Methylation Sensitive Restriction Enzyme (MSRE) method, which utilizes MSRE (such as HhaI, hpaII, aciI, etc.) characteristic of cutting only unmethylated recognition sequence, digests unmethylated DNA by enzyme digestion, and enriches methylated fragments. The method avoids chemical injury, but has inherent limitations of limited coverage, incomplete background digestion and single traditional MSRE-qPCR method. Other technologies, such as methods based on enrichment of methylation binding proteins, emerging enzymatic conversion methods (e.g. APOBEC enzymatic conversion), etc., or problems of high cost, complex operation, immature technology, etc., are also difficult to be used for multiplex methylation detection of low abundance cfDNA in clinical popularization. Therefore, development of a novel cfDNA methylation detection method construction strategy is needed, which can reserve and utilize limited cfDNA templates to the greatest extent, and realize synchronous, sensitive and quantitative detection of a plurality of target gene methylation sites through efficient enrichment and labeling means, so as to be suitable for clinical practical application scenes such as liquid biopsy. Disclosure of Invention Aiming at the defects and actual demands of the prior art, the invention provides a construction method of a nucleic acid sequence for cfDNA multiplex gene methylation detection and application thereof, which realizes negative screening of a non-methylated sequence through the cutting action of methylation sensitive restriction endonuclease on the non-methylated site, and simultaneously realizes specific enrichment and marking of the methylated sequence by utilizing a specific sticky end connection joint generated by enzyme cutting. In order to achieve the aim of the invention, the invention adopts the following technical scheme: In a first aspect, the invention provides a construction method of a nucleic acid sequence for cfDNA multiplex gene methylation detection, which comprises the steps of performing enzyme digestion on cfDNA to be detected by using methylation sensitive restriction enzyme to obtain enzyme fragments, connecting a linker to the cut ends of the enzyme fragments by using ligase to form linker products, and performing PCR amplification by using the linker products as templates to construct a recombinant nucleic acid sequence for target gene methylation site detection. The invention adopts non-methylation joints (a first joint and a second joint) to analyze methylation sites and construct a methylation detection method, wherein the non-methylation joints are subjected to restriction enzyme digestion by methylation sensitive restriction enzymes to generate sticky ends, and the sticky ends are introduced by using ligase. Methylation sensitive restriction enzymes recognize specific sites containing methylated C bases and cleave flanking the recognition site. Unmethylated linkers utilize unmethylated related cohesive end-producing endonucleases and include cohesive end-joining using methylation-sensitive restriction endonucleases. Methylation sensitive restriction enzymes recognize and cleave specific sites containing unmethylated C bases