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Preparation method of fusion gene positive control standard

A technology of fusion gene and positive control, applied in the field of biomedicine, can solve the problems of long production cycle, high cost, inflexible replacement of reference genes, etc., and achieve the effects of flexible use, cost saving, easy quantification and preservation

Active Publication Date: 2019-06-04
THE SECOND HOSPITAL OF DALIAN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, it is commonly used to clone the reference gene and the gene to be tested into plasmids in equal proportions to make internal controls, but this method has disadvantages such as high cost, long production cycle, and inflexibility in replacing the reference gene.

Method used

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  • Preparation method of fusion gene positive control standard
  • Preparation method of fusion gene positive control standard
  • Preparation method of fusion gene positive control standard

Examples

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preparation example Construction

[0032] A kind of preparation method of fusion gene positive control standard substance of the present invention comprises the following steps:

[0033] (1) extracting the total RNA in the expression sample of the fusion partner gene, and synthesizing cDNA by reverse transcription;

[0034] (2) Using the cDNA synthesized in step (1) as a template, and using gene-specific primers and gene junction primers as primers, amplify the upstream fusion partner gene and the downstream fusion partner gene of the fusion gene respectively by asymmetric PCR; wherein, for The molar concentration ratio of gene-specific primers and gene-junction primers for amplification of upstream fusion partner genes is 5-20:1, and the molar concentration ratio of gene-specific primers and gene-junction primers for amplification of downstream fusion partner genes is 5-20: 1;

[0035] (3) Mixing the PCR amplification products of the upstream fusion partner gene and the downstream fusion partner gene respecti...

Embodiment 1

[0051] Example 1 Non-small cell lung cancer ALK, preparation of ROS1 and RET fusion gene positive control standard

[0052] 1. According to the database (COSMIC v85), screen the ALK, ROS1 and RET fusion gene types and gene information reported in lung cancer, the fusion genes include: EML4(2)-ALK(20), EML4(3)-ALK(20 ), EML4(10)-ALK(20), EML4(14)-ALK(20), EML4(15)-ALK(20), EML4(17)-ALK(20), EML4(18)-ALK(20 ), EML4(20)-ALK(20), STRN(3)-ALK(20), TFG(4)-ALK(20), KLC1(9)-ALK(20), KLC1(10)-ALK(20 ), HIP1(21)-ALK(20), HIP1(28)-ALK(20), HIP1(30)-ALK(20), KIF5B(15)-ALK(20), KIF5B(17)-ALK(20 ), KIF5B(24)-ALK(20), CD74(6)-ROS1(32), CD74(6)-ROS1(34), EZR(10)-ROS1(34), TPM3(8)-ROS1(35 ), LRIG3(16)-ROS1(35), SLC34A2(4)-ROS1(32), SLC34A2(13)-ROS1(32), GOPC(4)-ROS1(36), SDC4(2)-ROS1(32 ), SDC4(2)-ROS1(34), SDC4(4)-ROS1(32), CCDC6(11)-RET(12), KIF5B(15)-RET(12), KIF5B(16)-RET(12 ), KIF5B(22)-RET(12), KIF5B(23)-RET(12), KIF5B(15)-RET(11), KIF5B(24)-RET(11).

[0053] Since the fusion genes ...

Embodiment 2

[0079] Example 2 Preparation and Application of EGFR Copy Number Analysis Internal Control Standard CFTR(7)-EGFR(20) Fusion Gene

[0080] 1. According to the literature, select the relatively conserved segment sequence of the CFTR gene on the same chromosome as the EGFR gene and on different chromosome arms as the reference gene for the analysis of the copy number of the EGFR gene. Using the HCT116 cell cDNA synthesized in Example 1 as a template, CFTR and EGFR gene fragments were amplified by asymmetric PCR respectively, and the primers used were SEQ ID NO:21~SEQ ID NO:22 and SEQ ID NO:95~SEQ ID NO:96 , amplification method, system, and conditions are the same as in Example 1.

[0081] 2. The self-annealing PCR reaction system and reaction conditions are the same as in Example 1.

[0082] 3. the self-annealing PCR product that obtains in above-mentioned steps 2 is diluted 1000 times and does template, and nested PCR amplifies the target fusion gene fragment, and primer seque...

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Abstract

The invention discloses a fusion gene positive control standard and a preparation method thereof. A fusion partner gene is amplified by a non-fusion gene positive sample, and the fusion partner gene nucleic acid fragment is ligated by asymmetric PCR combined with a self-annealing PCR method to obtain the corresponding fusion gene. Nested PCR and agarose gel electrophoresis are carried out for purification and recovery. After concentration determination, the corresponding fusion gene positive control standard is prepared. The fusion gene positive control standard and the preparation method thereof solve the problems that the fusion gene nucleic acid sequence positive control standard is difficult to obtain and the artificial synthesis cost is high. The fusion gene positive control standardhas application prospects in clinical fusion gene detection to assist diagnosis, treatment and prognosis. In addition, the synthetic fusion gene strictly controls the concentration of the two-side partner gene to 1:1, and can be used as an internal control for quantitative analysis such as gene copy number analysis. The fusion gene positive control product prepared by the preparation method has the advantages of simple preparation, flexibility, low cost and the like, and is of great significance in clinical detection, related quality inspection, and the like.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a fusion gene standard product and a preparation method thereof. Background technique [0002] Fusion gene is formed by breaking and abnormal splicing of two non-adjacent genes caused by chromosomal translocation, inversion or internal deletion. Fusion genes not only play an important role in the occurrence and evolution of malignant tumors of the lymphoid hematopoietic system, but also have been found in more and more solid tumors such as mesenchymal connective tissue tumors, prostate cancer, and lung cancer. Some specific fusion genes have been used clinically as important indicators for diagnosis, differential diagnosis, guiding medication and judging prognosis. Therefore, the clinical demand for tumor cell fusion gene detection is increasing. At present, the main methods of clinical fusion gene detection in China include immunohistochemical method, fluoresce...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12N15/11C12Q1/6851
Inventor 李梅吕申
Owner THE SECOND HOSPITAL OF DALIAN MEDICAL UNIV
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