Method for detecting variation and methylation of tumor specific genes in ctDNA

Abstract

The invention discloses a method for detecting variation and methylation of tumor specific genes in ctDNA (complementary deoxyribonucleic acid). According to the method, variation (including point mutation, insertion and deletion mutation, HBV integration and other mutation forms) and / or methylation of tumor specific genes in ctDNA can be detected in one sample at the same time, the sample size requirement is low, and an MC library prepared through the method can support 10-20 times of subsequent detection, the result of each detection can represent mutation conditions of all original ctDNA specimens and methylation modification conditions of enzyme cutting site coverage areas, and sensitivity and specificity cannot be reduced. The method has important clinical significance for early tumorscreening, disease tracking, curative effect evaluation, prognosis prediction and the like, and has important 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...

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

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