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FGFR1 gene mutation detection specific primer and liquid chip

A detection solution and specificity technology, which is applied in the field of molecular biology, can solve the problems that cannot meet the practical application and cannot be used, and achieve the effect of improving detection accuracy, consistent detection effect, and avoiding uncertain factors

Active Publication Date: 2013-04-10
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The PCR-RFLP method is based on the change of the restriction endonuclease recognition site caused by gene mutation, such as the loss of the site or the generation of a new site, a specific fragment is amplified by PCR, and then cut and amplified with a restriction endonuclease. Amplify the product and observe the size of the fragment by electrophoresis. This method is used to detect gene mutations with altered restriction sites, and can directly determine the genotype. However, this method cannot be used for the detection of gene mutations without new restriction sites.
Again, the above methods all have limitations in detection throughput, and can only detect one mutation type at a time, which cannot meet the needs of practical applications.

Method used

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  • FGFR1 gene mutation detection specific primer and liquid chip
  • FGFR1 gene mutation detection specific primer and liquid chip
  • FGFR1 gene mutation detection specific primer and liquid chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1 FGFR1 gene mutation detection liquid chip mainly includes:

[0021] 1. ASPE Primers

[0022] Specific primer sequences were designed for wild type and mutant type of two common genotypes C374T and C754A of FGFR1 gene. ASPE primers consist of "tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0023] The ASPE primer sequence (tag sequence+specific primer sequence) of table 1FGFR1 gene

[0024]

[0025] Each ASPE primer includes two parts, the 5' end is a specific tag sequence for the anti-tag sequence on the corresponding microsphere, and the 3' end is a mutant or wild-type specific primer fragment (as shown in the above table 1). All ASPE primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. Each synthesized primer was prepared into a stock solution of 100 pmol / mL with 10 mmol / L Tris Buffer.

[0026] 2. Microspheres coated with anti-tag sequences

[0027] According to th...

Embodiment 2

[0037] Example 2 Detection of samples using the FGFR1 gene mutation detection liquid chip described in Example 1

[0038] The formula of described various solutions is as follows:

[0039] 50mM MES buffer (pH5.0) formula (250ml):

[0040]

[0041] 2×Tm hybridization buffer

[0042] Reagent

[0043] Store at 4°C after filtration.

[0044] ExoSAP-IT kit was purchased from US USB Company.

[0045] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0046] 1. Sample DNA extraction:

[0047] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0048] 2. PCR amplification of samples to be tested

[0049] Design 2 pairs of primers and multiplex PCR to amplify 2 target sequences containing two common genotypes C374T and C754A of the FGFR1 gene in one step. The product sizes are 322bp and 403bp respectively. The primer sequences (SEQ ID NO.13-16) are listed above T...

Embodiment 3

[0083] The detection of the FGFR1 gene mutation site by the liquid chip of embodiment 3 different ASPE primers

[0084] 1. Design of liquid phase chip preparation (selection of tag sequence and Anti-tag sequence)

[0085] Taking the FGFR1 gene C754A site mutation detection liquid chip as an example, the specific primer sequence of the 3' end of the ASPE primer was designed for the wild type and mutant type of C754A, and the tag sequence of the 5' end of the ASPE primer was selected from SEQ ID NO.1 - SEQ ID NO.4, correspondingly, the anti-tag sequence coated on the microspheres and complementary to the corresponding tag sequence is selected from SEQ ID NO.9-SEQ ID NO.12. The specific design is shown in the following table (Table 6). The synthesis of ASPE primers, microspheres coated with anti-tag sequences, amplification primers, detection methods, etc. are as described in Example 1 and Example 2.

[0086] Table 6 Design of liquid phase chip preparation

[0087]

[0088]...

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Abstract

The invention discloses an FGFR1 gene mutation detection specific primer and a liquid chip. The liquid chip mainly comprises ASPE primers consisting of tag sequences at a 5' end and specific primer sequences aiming at target gene mutation sites at a 3' end, microspheres coated with anti-tag sequences and an amplification primer, wherein the specific primer sequences are as follows: SEQ ID NO.5 and SEQ ID NO.6 which aim at a C374T site, and / or SEQ ID NO.7 and SEQ ID NO.8 which aim at a C754A site. The detection liquid chip provided by the invention has the advantages that the matching rate between the detection result and a sequencing method is up to 100%, and the wild-type and mutant parallel detection of multiple mutant sites can be realized.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a specific primer for detecting FGFR1 gene mutation and a liquid phase chip. Background technique [0002] Fibroblast growth factors (fibroblast growth factors, FGFs) are a family of polypeptides with similar structures, and act through the transmembrane high-affinity fibroblast growth factor receptors (fibroblast growth factor receptors, FGFRs). FGFR belongs to receptor-type protein tyrosine kinases, and this family mainly includes four members: FGFR1, FGFR2, FGFR3 and FGFR4. FGFR is involved in the regulation of cell proliferation, apoptosis, migration, angiogenesis and many other processes. Because of their broad range of roles, FGFRs and other RTKs are normally tightly regulated. In tumors, such as breast cancer, bladder cancer, prostate cancer, etc., FGFR activating mutations or ligand / receptor overexpression lead to its continuous ac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 许嘉森吴诗扬
Owner SUREXAM BIO TECH
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