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Fusion gene nucleic acid detection method based on combination of capillary electrophoresis fragment analysis and first-generation sequencing

A technology of capillary electrophoresis and gene fusion, applied in biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc., can solve problems such as inability to obtain sequence information and cumbersome operation steps

Pending Publication Date: 2021-03-26
BEIJING SEARCH BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using capillary fragment analysis alone can only obtain the fragment size, but not the specific sequence information; while the next generation sequencing method can obtain the sequence, but usually only for a single nucleic acid fragment target, and the operation steps are cumbersome

Method used

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  • Fusion gene nucleic acid detection method based on combination of capillary electrophoresis fragment analysis and first-generation sequencing
  • Fusion gene nucleic acid detection method based on combination of capillary electrophoresis fragment analysis and first-generation sequencing
  • Fusion gene nucleic acid detection method based on combination of capillary electrophoresis fragment analysis and first-generation sequencing

Examples

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Effect test

Embodiment 1

[0068] Example 1. Fusion gene nucleic acid detection method based on the combination of capillary electrophoresis fragment analysis and first-generation sequencing 1. Design of multiple amplification primers

[0069] According to the connection sequence of multiple target fusion genes (the sequence containing the connection site of the fusion gene is 100-300bp in size) as the target fragment, set multiple pairs of amplification primers for multiple nucleic acid detection, and each pair of amplification primers is specific Amplify 1 target fusion gene:

[0070] Each pair of amplification primers consists of primer A and primer B, wherein:

[0071] Starting from the 5' end, the primer A is sequentially composed of an adapter sequence A for binding the sequencing primer and a specific sequence A specifically binding to the target fragment, and the 5' end of the primer A is fluorescently modified. Among them, the fluorescently modified groups can be, for example, FAM, HEX, VIC, T...

Embodiment 2

[0102] Example 2. Application of fusion gene nucleic acid detection method based on the combination of capillary electrophoresis fragment analysis and generation sequencing

[0103] 1. Design of Multiplex Amplification Primers

[0104] Amplification primer pairs were designed for the four common fusion genes KMT2A-AFF1, BCR-ABL1, CBFB-MYH11 and TCF3-PBX1 in leukemia.

[0105] Select the junction sequence of each fusion gene (the size of the junction site containing the fusion gene is within the sequence of 200bp) as the target fragment, and set up multiple pairs of amplification primer pairs (shown in Table 1) for multiple nucleic acid detection respectively. The primer pair specifically amplifies a target fusion gene:

[0106] Among them, the upstream primer of KMT2A-AFF1 is marked with FAM (the corresponding color is blue), the downstream primer of BCR-ABL1 is marked with HEX (the corresponding color is green), and the upstream primer of CBFB-MYH11 is marked with TAMRA (the...

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Abstract

The invention discloses a fusion gene nucleic acid detection method based on combination of capillary electrophoresis fragment analysis and first-generation sequencing. The invention provides a kit for detecting multiple fusion genes. The kit comprises multiple pairs of amplification primer pairs, wherein each pair of amplification primers consists of a primer A and a primer B; the primer A sequentially consists of a linker sequence A used for combining a sequencing primer and a specific sequence A specifically combined with a target sequence of the fusion gene from the 5' end; the primer B sequentially consists of a linker sequence B for adjusting the length of an amplification product and a specific sequence B specifically combined with a target sequence of the fusion gene from the 5' end; and the 5' end of the primer A is modified with a fluorophore. By combining the capillary electrophoresis fragment analysis and the first-generation sequencing, fusion gene nucleic acid-based multiple detection and sequence synchronous acquisition are realized, so that while fragment analysis-based multiple detection is performed, first-generation sequencing is performed on a positive fusion gene to obtain a specific sequence of the positive fusion gene.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a fusion gene nucleic acid detection method based on the combination of capillary electrophoresis fragment analysis and first-generation sequencing. Background technique [0002] Fusion genes formed by chromosomal mutations usually play a key driving role in the occurrence and progression of tumors. More and more studies have shown that the detection and identification of fusion genes is of great importance for the diagnosis, treatment, prognosis evaluation and subsequent diagnosis of tumors. Lesion residual disease monitoring is of great significance. Current methods for detecting fusion genes usually include fluorescence in situ hybridization (FISH), real-time fluorescent PCR and next-generation high-throughput sequencing, etc., but these methods have their own defects or disadvantages. FISH technology has the characteristics of complicated operation, high labor cost a...

Claims

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Application Information

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IPC IPC(8): C12Q1/6827C12Q1/6869C12N15/11
CPCC12Q1/6827C12Q1/6869
Inventor 刘金超刘孔尚刘明坤叶锋
Owner BEIJING SEARCH BIOTECH
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