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IL-23R gene polymorphism detection specific primer and liquid phase chip

A technology of IL-23R and gene polymorphism, applied in the field of molecular biology, can solve problems that cannot meet practical applications, achieve good signal-to-noise ratio, consistent detection effect, and simple steps

Inactive Publication Date: 2011-08-10
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, TaqMan technology can only add one fluorescent detection probe to detect one genotype in one tube reaction. If it is necessary to detect the wild type and mutant type of a gene, it is necessary to separate two tube reactions and add the wild type probe respectively. Two independent assays for needle and mutant probes
Other detection methods also have similar detection throughput limitations, and can only detect one mutation type at a time, which cannot meet the needs of practical applications.

Method used

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  • IL-23R gene polymorphism detection specific primer and liquid phase chip
  • IL-23R gene polymorphism detection specific primer and liquid phase chip
  • IL-23R gene polymorphism detection specific primer and liquid phase chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 IL-23R gene polymorphism detection liquid chip mainly includes:

[0024] 1. ASPE Primers

[0025] Specific primer sequences were designed for wild-type and mutant types of four common genotypes C214T, C235T, T82G and C53T of IL-23R gene. ASPE primers consist of "Tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0026] The ASPE primer sequence (Tag sequence+specific primer sequence) of table 1 IL-23R gene

[0027]

[0028]

[0029] 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.

[0030] 2. Microspheres coated with anti-tag sequ...

Embodiment 2

[0043] Example 2 Detection of samples using IL-23R gene detection liquid chip

[0044] The formula of described various solutions is as follows:

[0045] 50mM MES buffer (pH5.0) formula (250ml):

[0046]

[0047] 2×Tm hybridization buffer

[0048] Reagent

[0049] Store at 4°C after filtration.

[0050] ExoSAP-IT kit was purchased from US USB Company.

[0051] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0052] 1. Sample DNA extraction:

[0053] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0054] 2. PCR amplification of samples to be tested

[0055] Four pairs of primers were designed, and multiplex PCR amplified four target sequences containing four common genotypes C214T, C235T, T82G and C53T of the IL-23R gene in one step. The product sizes were 412bp, 480bp, 345bp and 343bp, respectively. SEQID NO.25-32) are shown in the above Table 3. ...

Embodiment 3

[0097] Example 3 Detection of IL-23R gene polymorphism site by liquid chip with different ASPE primers

[0098] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0099]Taking the IL-23R gene C235T and T82G 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 C235T and T82G, and the Tag sequence at the 5' end of the ASPE primer was selected. From SEQ ID NO.1-SEQ ID NO.8, correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.17-SEQ ID NO.24. The specific design is shown in the following table (Table 7). 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.

[0100] Table 7 Design of liquid phase chip preparation

[0101] ...

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Abstract

The invention discloses an interleukin-23 receptor (IL-23R) gene polymorphism detection specific primer and an IL-23R gene polymorphism detection liquid phase chip. The liquid phase chip mainly comprises an ASPE primer, microspheres and an amplified primer, wherein the ASPE primer consists of a tag sequence of a 5' end and specific primers of a 3' end, and the specific primers are selected from more than one pair of SEQ ID NO.9 and SEQ ID NO.10 aiming at C214T, SEQ ID NO.11 and SEQ ID NO.12 aiming at C235T, SEQ ID NO.13 and SEQ ID NO.14 aiming at T82G, and SEQ ID NO.15 and SEQ ID NO.16 aimingat C53T. The IL-23R gene polymorphism detection liquid phase chip has very good signal-noise ratio, the designed probe and an anti-tag sequence basically have no cross reaction, and the selection of a tag sequence and the anti-tag sequence and the combination of the tag sequence and the specific ASPE primer can avoid cross reaction and realize parallel detection of multiple polymorphic sites.

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 IL-23R gene polymorphism and a liquid phase chip. Background technique [0002] Human interleukin-23 receptor (hIL-23R, IL-23R) is a heterodimeric cytokine composed of IL-23p19 and IL-12p40, which is mainly composed of activated dendritic cells and giant cells. Secreted by phagocytes, it is a member of the IL-12 inflammatory factor family and transmits active signals through the Jak / Stat pathway. The coding gene of IL-23R is located at the position of 1p31.2~32.1 on chromosome 1, and the coding region contains 29kb bases and consists of 11 exons. [0003] Studies have shown that the gene polymorphism of IL-23R is related to the immune response and the formation of various lung diseases, such as: tuberculosis, Klebsiella pneumoniae pneumonia, whooping cough and other infectious diseases and pulmonary fibrosis;...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 许嘉森郭婧刘志明
Owner SUREXAM BIO TECH
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