Enrichment method for peripheral blood free tumor DNA, kit and application thereof

Abstract

The invention discloses an enrichment method for peripheral blood free tumor DNA, a kit and application thereof. The enrichment method disclosed by the invention comprises the following steps: capturing a tumor DNA fragment from a cfDNA library by adopting a PAP probe, and performing amplification by taking the PAP probe as a primer and taking the captured tumor DNA fragment as a template, whereinthe PAP probe comprises generic primer sequences and specific recognition sequences from the end 5' to the end 3'; the end 3' of the specific recognition sequence comprises at least 30 bp of sequences which are hybridized and complemented with the tumor DNA fragment; the terminus 3' of the PAP probe is dideoxy nucleotide. According to the enrichment method disclosed by the invention, the PAP probe is used for capturing the target tumor DNA fragment, and extension is carried out by using the PAP probe; the PAP probe is high in specificity and high in capturing efficiency; the purity of an enriched product is guaranteed, and a guarantee is provided for subsequent sequencing. The enrichment method disclosed by the invention provides a novel free tumor DNA enrichment scheme for clinical detection of cfDNA.

Description

technical field [0001] The present application relates to the field of tumor detection, in particular to a method for enriching free tumor DNA in peripheral blood, a kit and its application. Background technique [0002] The occurrence of malignant tumors is the result of accumulation of multiple genes and multiple events. In the process of tumor clonal evolution, a series of gene mutations are often accumulated, which may involve changes in multiple genes on different chromosomes, such as oncogenes, tumor suppressor genes, apoptosis genes, cell cycle regulatory genes and maintenance of cell genome stability sex genes, etc. High-throughput sequencing technology can be used to study the mutations and changes in expression levels of the above-mentioned genes and their expressed mRNAs, thereby revealing changes in signaling and metabolic pathways in tumorigenesis and evolution. [0003] The understanding of tumor mechanism and the development of science and technology have fu...

AI Technical Summary

Problems solved by technology

[0007] Using peripheral blood cell-free tumor DNA (abbreviated ctDNA) to detect multi-target genes in lung cancer can solve two major clinical problems: (1) the primary tumor recurs, and tissue biopsy is difficult both ethically and practically. And it may delay treatment and lead to complications; (2) single-point biopsy can only represent the pathological conditions of tumors at selected sites, while ctDNA can represent the overall genetic information of multiple lesions

[0009] The above four types of target region enrichment methods, among which, the enrichment method based on probe hybridization has disadvantages such as long experimental cycle, low capture efficiency, and high cost.

The methods of multiplex PCR and circular PCR do not support or have low support for gene fusion mutations

Extended capture of a probe in the same direction can support gene fusion mutation, but there are disadvantages such as long capture time, insufficient capture efficiency or high cost, resulting in prolonged delivery time, loss of effective data and high product cost

The above defects limit the application of traditional target region enrichment methods in clinical detection, especially in the field of liquid biopsy

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