Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

A method for detecting mutation and methylation of tumor-specific genes in ctDNA

A technology of methylation and DNA molecules, applied in the field of biomedicine, can solve the problems of extended detection cycle, low sensitivity of low-frequency mutation detection, low ctDNA mutation content, etc., and achieve the effect of low sample size requirement and great application value

Active Publication Date: 2022-03-22
CANCER INST & HOSPITAL CHINESE ACADEMY OF MEDICAL SCI +1
View PDF17 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main difficulties are as follows: 1) The amount of ctDNA samples obtained from one blood draw is limited, usually only enough to support 1-2 tests, which results in the fact that ctDNA clinical tests are usually single-platform and one-time, and it is difficult to achieve simultaneous detection in one sample Mutation detection and methylation detection, especially the methylation detection technology that relies on bisulfite conversion, will cause more DNA loss during the process; 2) the bisulfite conversion step of the methylation detection technology, It will cause the DNA sequence to fail to present most of the mutation information, and the loss of information carried by this part of DNA may lead to a decrease in the detection sensitivity of low-frequency mutations; 3) In clinical detection, it is often necessary to judge the target and plan of subsequent detection based on the results of the first detection, which is Blood needs to be drawn again in follow-up testing, and the testing cycle is prolonged; in addition, ctDNA-related clinical testing or research often needs to compare the pros and cons of multiple technologies, which requires several times the normal blood volume, which is usually unacceptable to patients; 4) Regardless of the PCR method or the capture method, the noise mutations generated during the amplification process will seriously interfere with the detection of low-frequency mutations in ctDNA, resulting in false positive results and misleading the diagnosis and treatment of patients; Contamination occurs, causing false positive results

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A method for detecting mutation and methylation of tumor-specific genes in ctDNA
  • A method for detecting mutation and methylation of tumor-specific genes in ctDNA
  • A method for detecting mutation and methylation of tumor-specific genes in ctDNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0119] Embodiment 1, the construction of MC library

[0120] 1. Methylation-sensitive restriction endonuclease digestion

[0121] Take 5-40ng cfDNA, configure the reaction system as shown in Table 1, and then carry out enzyme digestion treatment in a PCR instrument according to the procedures in Table 2 to obtain enzyme digestion products (stored at 4°C).

[0122] Both Restriction Enzyme and Restriction Enzyme 10×Buffer are products of ThermoFisher. Restriction Enzyme and Restriction Enzyme 10×Buffer can be selected according to different target regions to be tested, and the selection criterion is that the region to be tested contains at least one cleavage site of the methylation-sensitive restriction endonuclease.

[0123] Table 1. Reaction system

[0124] Element volume cfDNA 16.8μl Restriction Enzyme 10×Buffer 2μl Acetylated BSA (10μg / μl concentration) 0.2μl Restriction Enzyme (10U / μl concentration) 1μl total capacity 20μl ...

Embodiment 2

[0165] Example 2, RaceSeq enriches the target region and constructs a sequencing library

[0166] Such as figure 2 As shown, using primers designed for the relevant regions of Chinese hepatocellular carcinoma highly mutated genes (TP53, CTNNB1, AXIN1, TERT), HBV integration hotspot regions and specific hypermethylated regions of hepatocellular carcinoma (EMX1, LRRC4, BDH1, etc.), Cooperate with the fixed primers to perform two rounds of PCR amplification on the MC library, and the amplified product is the sequencing library.

[0167] figure 2 Among them, a is the upstream primer of the first round of library amplification, b is the upstream primer of the second round of library amplification, c is the downstream primer library of the first round of library amplification, which is used for the enrichment of specific target sequences, and d is the second round of library amplification Amplify the downstream primer library for the enrichment of specific target sequences, e is...

Embodiment 3

[0212] Example 3, capture and sequencing of MC library

[0213] Such as image 3 As shown, the enrichment of the target region can be captured based on the optimized design of the existing commercial target capture kit. For example: for capture based on methylated regions, please refer to Roche SeqCap Epi CpGiant Enrichment Kit (Roche07138881001) or Illumina Infinium Methylation EPIC BeadChipWG-317-1001), and the design of targeted capture of methylated regions needs to be carried out according to the degree of coverage of restriction sites Screen and adjust probes for bases converted based on bisulfite treatment. The capture based on the gene variation region can refer to the Agilent sureselect XT target capture kit (Agilent5190-8646), and only the primers in the last step of PCR amplification are replaced with the following primers:

[0214] The upstream primer is: 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCT ACACGACGCTCTTCCG AT CT-3' (sequence 403) ( image 3 In "a"), t...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a method for detecting the variation and methylation of tumor-specific genes in ctDNA. The method can simultaneously detect the variation of tumor-specific genes in ctDNA (including point mutations, insertion-deletion mutations, and HBV integration) in one sample. and / or methylation method, not only the sample size requirement is low, but also the MC library prepared by this method can support 10‑20 follow-up detections, and the results of each detection can represent all original ctDNA samples The mutation status and the methylation modification of the region covered by the restriction site will not cause a decrease in sensitivity and specificity. The invention has important clinical significance for early tumor screening, disease tracking, curative effect evaluation, prognosis prediction, etc., and has great application value.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a method for detecting the variation and methylation of tumor-specific genes in ctDNA. Background technique [0002] Circulating tumor DNA (ctDNA) is derived from DNA fragments produced by apoptosis, necrosis or secretion of tumor cells, and contains the same gene variation and epigenetic modification as tumor tissue DNA, such as point mutation, gene rearrangement, fusion, Copy number variation, methylation modification, etc. The detection of ctDNA can be applied to various aspects such as early screening of cancer, diagnosis and staging, guidance of targeted drugs, efficacy evaluation, and recurrence monitoring. Combining the mutation and methylation information of tumor-specific genes carried by ctDNA can help improve the sensitivity and specificity of detection, and detect cancer traces earlier, which is of great significance for early screening of tumors. [0003] Exi...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C40B50/06C40B40/06C12Q1/6886C12Q1/6806C12Q1/6869C12N15/11
CPCC40B50/06C40B40/06C12Q1/6886C12Q1/6806C12Q1/6869C12Q2600/154C12Q2600/156C12Q2600/118C12Q2525/191C12Q2521/301C12N15/1093C12Q2525/155C12Q2525/30C12Q2531/107C12Q2537/143C12N15/1034C40B50/00C12N15/1065C12Q1/6844
Inventor 焦宇辰曲春枫王宇婷王沛陈坤宋欠欠刘慧王京京王思振阎海
Owner CANCER INST & HOSPITAL CHINESE ACADEMY OF MEDICAL SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products