Method for detecting whether fusion mutation of target gene occurs, primer combination, kit and application thereof

A fusion mutation and primer combination technology, applied in the fields of biochemical equipment and methods, combinatorial chemistry, recombinant DNA technology, etc., can solve the problems of low capture efficiency, false negatives, difficult and unknown breakpoint fusion gene detection, etc., and achieve the enrichment effect. Good results

Active Publication Date: 2019-01-18
SIMCERE DIAGNOSTICS CO LTD +1
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AI Technical Summary

Problems solved by technology

[0005] Despite many advantages, the traditional amplicon sequencing method still has many deficiencies in the detection of gene fusions. The specific performance is as follows: (1) Due to the inability to obtain the partner gene information and breakpoint position of the unknown fusion gene, the traditional amplicon sequencing method Sequencing methods are difficult to detect fusion genes with unknown breakpoints. In the prior art, there are few kits based on amplicon sequen

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  • Method for detecting whether fusion mutation of target gene occurs, primer combination, kit and application thereof
  • Method for detecting whether fusion mutation of target gene occurs, primer combination, kit and application thereof
  • Method for detecting whether fusion mutation of target gene occurs, primer combination, kit and application thereof

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Embodiment 1

[0061] This embodiment designs primer combinations that can be used to detect ALK fusion gene, NTRK1 fusion gene, RET fusion gene, ROS1 fusion gene, FGFR3 fusion gene and NTRK3 fusion gene, and its design principles are as follows:

[0062] (1) Select a target gene that may be fused with other genes, and determine the DNA region on the target gene that may be broken and fused;

[0063] (2) predicting the relative positional relationship between the other genes and the target gene after the cleavage fusion occurs, and designing a primer set according to the relative positional relationship:

[0064] a. If after fusion, the other genes are located at the 5' end of the target gene, then sequentially design multiple 3' ends and the sense strand along the direction from the 3' end to the 5' end of the sense strand of the DNA region The primers with reverse complementary pairing of the strands constitute primer set 1, wherein, in each primer, the region that is reversely complementa...

Embodiment 2

[0073] The primer combination described in the above table 1 was used to establish the amplification library for sequencing. The specific library construction method is as follows, Figure 2 to Figure 13 The primer combination shown was outsourced and named as the library construction primer set. Figure 14 The general primers / upstream primers shown were synthesized outsourced, and other reagents used for library construction were purchased from Qiagen:

[0074] 1. Fragmentation, end repair and "A" ligation

[0075] 1.1. According to the sample type and sample quality, take 20-80ng of DNA for subsequent library construction operations. Among them, it is recommended to input 20-40ng for cfDNA samples and 40-80ng for FFPE samples. On ice, prepare fragmentation, end repair, and "A" ligation mixes according to Table 2. Centrifuge briefly, then pipette 7-8 times to mix and centrifuge briefly again.

[0076] Table 2 Fragmentation, end repair and "A" ligation reaction mix

[0077...

experiment example 1

[0130] In this experimental example, the performance of the optimized primer combination and library construction method finally optimized in Example 1 of the present invention was firstly verified by using the internationally recognized Horizon standard. Build a library and use Illumina's Nextseq550AR sequencer to sequence the constructed library and analyze the off-machine data, and count whether the detected mutation results are consistent with the results marked by the manufacturer. Secondly, in addition to standard products, we also verify the negative and positive clinical samples collected on the market and verified by other testing platforms, and count the consistency of the results. In addition, considering that there are few amplicon-based sequencing kits in the prior art to detect fusion mutations at the DNA level, this experimental example chooses a commercialized Qiagen non-specific gene mutation detection kit for fusion mutations (Lung Cancer Panel), as a control...

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Abstract

The invention relates to a method for detecting whether fusion mutation of target gene occurs, a primer combination, a kit and an application thereof. A plurality of primers are sequentially designedaccording to the possible gene fusion occurrence direction by the primer design method of the invention, forming a primer combination, wherein the primers of the primer combination extend along the direction in which gene fusion occurs, can carry out directional enrichment on the target gene in which unknown fusion occurs, overcomes the disadvantage that the existing amplicon sequencing method cannot enrich the unknown fusion gene targetedly, and is conducive to the discovery and detection of the unknown fusion type of the gene; At the same time, the interval between two adjacent primers is about 0 - 130 bp, which is good for highly fragmented samples (such as cfDNA, FFPE samples). The invention also designs primer combinations for detecting fusion mutations of lung cancer genes (includingALK gene, NTRK1 gene, RET gene, ROS1 gene, FGFR3 gene and NTRK3 gene), After multiple rounds of detection and optimization of the primer sequence, the homogeneity of the sequencing data was more than85%, and the capture efficiency was more than 92%. Finally, the effective detection of unknown fusion mutation of various lung cancer genes was successfully achieved.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to a method for detecting whether a fusion mutation occurs in a target gene, a primer combination, a kit and an application thereof. Background technique [0002] Fusion gene is a kind of gene mutation, which refers to a new gene formed by the fusion of all or part of the sequences of two genes, which may be the result of chromosomal translocation, middle deletion or chromosomal inversion. In most cases, fusion genes can lead to the production of proteins with abnormal sequences or functions, or the dysregulation of the expression of certain genes, which can cause or promote the occurrence of tumors. Since the discovery of the Philadelphia chromosome in 1973, with the development of detection technology, especially the development of sequencing technology, scientists have successively found blood cancers such as leukemia and solid tumors such as lung cancer, prostate cancer, breast can...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C12Q1/6886C12N15/11C40B50/06
CPCC12Q1/6806C12Q1/6886C12Q2600/156C40B50/06
Inventor 张超李杜衡石亚辉陈斌任用
Owner SIMCERE DIAGNOSTICS CO LTD
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