A kind of enrichment method, kit and application of peripheral blood free tumor dna

Abstract

The application discloses a method for enriching free tumor DNA in peripheral blood, a kit and an application thereof. The enrichment method of the present application includes the use of PAP probes to capture tumor DNA fragments from the cfDNA library, and use the PAP probes as primers to amplify the captured tumor DNA fragments as templates; the PAP probes extend from the 5' end to the The 3' end is composed of a universal primer sequence and a specific recognition sequence, the 3' end of the specific recognition sequence includes at least 30 bp hybridized complementary sequence with the tumor DNA fragment, and the 3' end of the PAP probe is a dideoxy nucleotide. The enrichment method of this application uses PAP probes to capture target tumor DNA fragments, and uses PAP probes to extend; it has good specificity and high capture efficiency; it ensures the purity of enriched products and provides a guarantee for subsequent sequencing . The enrichment method of this application provides a new free tumor DNA enrichment scheme for the clinical detection of cfDNA.

Description

Technical field [0001] The present application relates to the field of tumor detection, and in particular, a enrichment method, kit, and application thereof, related to peripheral hemorrhagic tumor DNA. Background technique [0002] The occurrence of malignant tumors is a result of a multi-gene income accumulation. In the process of tumor cloning, often accumulate a series of genetic mutations, may involve variations in various genes on different chromosomes, such as carcinoid genes, tumor suppressor genes, apoptotic genes, cell cycle regulatory genes and maintain cell genome stability. Sex gene, etc. The above gene and its expression of mutation and expression levels thereof can be studied by high-throughput sequencing techniques, thereby disclosing changes in tumor generation and evolution in tumors and evolution. [0003] The understanding of tumor mechanism and the development of science and technology have further promoted the transformation of tumor therapy concepts, and in...

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) primary tumor recurrence, tissue biopsy has certain difficulties both ethically and practically, And it may delay treatment and lead to complications; (2) single-point biopsy can only represent the pathological conditions of the tumor at the selected site, 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

Images