Method, probe library and kit for detecting tumor ctDNA methylation with high throughput and high sensitivity

A high-sensitivity, methylation technology, applied in the direction of recombinant DNA technology, biochemical equipment and methods, DNA / RNA fragments, etc., can solve the problems of limited flux, incomplete transformation, easy fragmentation of ctDNA, etc., to improve Sensitivity, increased sensitivity, and the effect of avoiding a large amount of DNA degradation

Active Publication Date: 2022-03-22
CHONGQING UNIV CANCER HOSPITAL
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Problems solved by technology

[0005]At present, bisulfite conversion is the most widely used ctDNA methylation research method, but after bisulfite treatment, ctDNA is easily fragmented and cannot be completely converted
In addition, previous technologies mostly use single PCR or multiple PCRs to detect a single site or a small number of methylated sites, and their throughput is limited

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  • Method, probe library and kit for detecting tumor ctDNA methylation with high throughput and high sensitivity
  • Method, probe library and kit for detecting tumor ctDNA methylation with high throughput and high sensitivity
  • Method, probe library and kit for detecting tumor ctDNA methylation with high throughput and high sensitivity

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Embodiment 1

[0027] The invention discloses a high-throughput and high-sensitivity method for detecting tumor ctDNA methylation. The technical flow diagram is as follows: figure 1 As shown, the specific examples are as follows:

[0028] 1. Preparation of DNA sample bank

[0029] 1. Blood cell-free DNA (cfDNA) extraction: Use Magen’s cell-free DNA extraction kit, collect fresh blood with EDTA-Na+ tubes, 7-8ml; centrifuge at room temperature (1000g / min, 10min), collect supernatant, and store at 4°C Centrifuge at 12,000 rpm for 10 min, and the obtained supernatant (4-5 ml) is subjected to cfDNA extraction and purification according to the kit instructions.

[0030] 2. cfDNA end repair: DNA fragments are subjected to end repair, which can be performed using Klenow fragments, T4 DNA polymerase and T4 polynucleotide kinase, wherein the Klenow fragments have 5'-3' polymerase activity and 3'-5 'polymerase activity, but lacks 5'-3' exonuclease activity. Thus, the end repair of the DNA fragment c...

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Abstract

The invention relates to the technical field of biology, and particularly discloses a method, a probe library and a kit for detecting tumor ctDNA methylation with high throughput and high sensitivity. The method comprises the following steps: introducing two common sequencing linker sequences into DNA of a blood sample to be detected, carrying out enzyme digestion by using methylation-insensitive incision enzyme, leaving methylated DNA molecules and a non-target region without enzyme digestion sites, then carrying out PCR amplification and probe hybridization to enrich the DNA of the target region, and carrying out sequencing on the DNA of the blood sample to be detected. The methylation state of a ctDNA target area in a blood sample to be detected is analyzed and identified through high-throughput sequencing, the method has the advantages of being high in sensitivity, high in throughput and capable of achieving digital analysis, bisulfite conversion is not needed, and the problems of fragmentation and incomplete conversion caused when the ctDNA is treated through bisulfite are effectively solved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for detecting DNA methylation with high throughput and high sensitivity. Background technique [0002] Cancer is a collective term for a series of malignant tumors and is one of the leading causes of human death worldwide. In 2020, there will be 9.96 million cancer deaths worldwide, of which 1.8 million will be lung cancer deaths, accounting for 18% of the total cancer deaths, and it is the cancer with the highest mortality rate in the world. The main treatment methods for cancer include surgery, radiotherapy, chemotherapy and molecular targeted therapy, etc. However, the average five-year survival rate of patients with advanced cancer is only 26%. However, early detection, early diagnosis, and early treatment of cancer patients usually have better treatment effects, with an average five-year survival rate of 91%, so early screening of tumors can effectively reduce mortalit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6869C12Q1/6813C12N15/11
CPCC12Q1/6886C12Q1/6869C12Q1/6813C12Q2600/154C12Q2531/113C12Q2535/122C12Q2525/191
Inventor 唐超施小龙吴永忠王颖綦俊
Owner CHONGQING UNIV CANCER HOSPITAL
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