DNA-based fusion gene quantitative sequencing library construction, detection method and its application

Abstract

The invention discloses a quantitative sequencing and library building method for a fusion gene on the basis of DNA (Deoxyribonucleic Acid). The quantitative sequencing and library building method comprises the following steps: firstly, constructing a genome fragmentation DNA library and purifying the library; secondly, capturing a fusion gene generation region by PCR (Polymerase Chain Reaction) amplification, purifying the captured gene and enriching a sequence containing a specific primer fragment; thirdly, capturing a target fragment containing the fusion gene by nested PCR amplification; fourthly, constructing a DNA sequencing library with high-throughput sequencing. The invention also discloses a quantitative sequencing and detecting method for the fusion gene by using the DNA sequencing library prepared by the quantitative sequencing and library building method, application of the quantitative sequencing and detecting method as well as a detection kit containing the DNA sequencing library. According to the quantitative sequencing and library building method disclosed by the invention, the downstream where a fusion breakpoint occurs is anchored by using a one-way specific primer; a target sequence is obtained by pairing specific primers with universal primers and using a PCR method; the background is further reduced label enriching and nested PCR, so that the specificity is improved, the time for building the library is shortened, and the cost for building the library is reduced; the quantitative sequencing and library building method is suitable for an FFPE (Formalin Fixed And Parafiin Embedded) sample or liquid biopsy.

Description

Technical field [0001] The invention relates to the field of gene detection, in particular to the sequencing and detection of fusion genes, and more specifically to the preparation of a DNA sequencing library of fusion genes, and a method for high-throughput sequencing detection of fusion genes using the DNA sequencing library. Background technique [0002] Since the first fusion gene (BCR / ABL1) was tested and confirmed in the early 1980s, the fusion gene has become an important marker for multiple cancers, for example: BCR / ABL1 is used to detect chronic myelogenous leukemia; EML4 / ALK is used Detection of malignant lung adenocarcinoma; TMPRSS2 / ERG is used to detect prostate cancer, etc. [0003] Since the beginning of the 21st century, with the rise of targeted drugs, cancer treatment drugs, especially non-small cell lung cancer (NSCLC) drugs, have also begun to use the metabolic pathways where fusion genes are located. For example, in 2011, crizotinib was approved by the FDA for ...

AI Technical Summary

Problems solved by technology

However, these early cytology and molecular biology techniques have the following defects for the detection of fusion genes: 1) the detection time is long; 2) the false positive rate is high; Conducive to standardization; 4) In the case of low content of fusion genotype cells in cancer tissue, it cannot be effectively detected

[0007] However, compared with ctDNA, the degree of fragmentation of ctRNA is more serious, generally at 10-25bp (the degree of fragmentation of ctDNA is generally at 150-200bp), therefore, ctRNA is not suitable for fusion gene under the existing technology detection, which makes RNA-based detection of fusion genes difficult to apply in liquid biopsies

[0008] In addition, RNA-based fusion gene detection also faces the problem that it is difficult for most patients to accept multiple puncture sampling, and only the remaining FFPE (formalin-fixed paraffin-embedded) samples for pathological testing can be obtained

The RNA in FFPE samples has been severely degraded due to formaldehyde fixation, and it is difficult to provide RNA fragments of sufficient length for detection

[0009] At present, the DNA-based fusion gene sequencing method is identified by capturing the region of the fusion gene with a probe in the genomic library. The advantage of this method is that it is purposeful and can save data and cost; When detecting free DNA in liquid biopsy, the effective data ratio is low, and it is necessary to increase the amount of initial DNA and sequencing data, resulting in high cost, long operation time, high difficulty in operation, and even affects the detection

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