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

Abstract

The invention discloses a DNA-based method for quantitatively sequencing a fusion gene library, the steps comprising: 1) genomic DNA fragmentation; 2) DNA fragment sorting; 3) unidirectional specific PCR amplification to obtain fusion sites Amplify the product; 4) connect the single-stranded universal library linker sequence at the 3' end of the amplified product sequence; 5) universal PCR amplification, enrich the fragments of the fusion gene region, and obtain a sequencing library after purification and quantification. The invention also discloses a method for constructing a DNA library by the above method for sequencing detection of fusion genes, the application of the method, and a set of unidirectional specific primers for fusion genes used in the above method for building a library. The invention amplifies DNA fragments specifically in one direction, captures fusion sequences, and then obtains a DNA library for NGS sequencing through enrichment, which not only improves the ratio of captured effective data, but also accelerates the speed of library construction and detection, and is suitable for FFPE samples or liquid biopsies.

Description

technical field [0001] The present invention relates to the field of gene detection, in particular to the sequencing 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 detected and confirmed in the early 1980s, the fusion gene has become an important marker of many cancer types, for example: BCR / ABL1 is used to detect chronic myelogenous leukemia; EML4 / ALK is used to detect Detection of malignant lung adenocarcinoma; TMPRSS2 / ERG for detection of prostate cancer, among others. [0003] Since the beginning of the 21st century, with the rise of targeted drugs, cancer treatment drugs, especially those for non-small cell lung cancer (NSCLC), have also begun to use the metabolic pathway where the fusion gene is located. For example, in 2011, cri...

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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