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tnf gene mutation detection specific primer and liquid phase chip

A detection solution and chip technology, applied in the field of molecular biology, can solve the problems of easy contamination of samples, high false positive rate, low sensitivity, etc., and achieve the effect of avoiding uncertain factors, low cross-reaction rate and good detection specificity

Active Publication Date: 2016-05-18
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.
Fluorescent quantitative PCR technology has the disadvantages of low sensitivity, easy sample contamination, and high false positive rate
The detection efficiency of SNaPShot technology is high, but there are disadvantages such as expensive instruments, high average price of sample analysis, high requirements for samples, and poor repeatability, and the research on the relationship between many diseases and susceptibility genes still needs small-scale research in conventional laboratories

Method used

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  • tnf gene mutation detection specific primer and liquid phase chip
  • tnf gene mutation detection specific primer and liquid phase chip
  • tnf gene mutation detection specific primer and liquid phase chip

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0018] Embodiment 1 TNF gene mutation detection liquid chip mainly includes:

[0019] 1. ASPE Primers

[0020] Specific primer sequences were designed for wild-type and mutant types of seven common genotypes of TNF gene G8226A, G-308A, A1031G, G863T, G857A, G8146C and C5478T. ASPE primers consist of "tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0021] Table 1 ASPE primer sequence of TNF gene (tag sequence + specific primer sequence)

[0022]

[0023]

[0024] Each ASPE primer consists of 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 Table 1 above). 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 / LTrisBuffer.

[0025] 2. Microspheres coated with...

Embodiment 2

[0038] Example 2 Detection of samples using the TNF gene mutation detection liquid chip described in Example 1

[0039] The formula of described various solutions is as follows:

[0040] 50mM MES buffer (pH5.0) formula (250ml):

[0041]

[0042] 2×Tm hybridization buffer

[0043]

[0044] Store at 4°C after filtration.

[0045] ExoSAP-IT kit was purchased from US USB Company.

[0046] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0047] 1. Sample DNA extraction:

[0048] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0049] 2. PCR amplification of samples to be tested

[0050] Design 4 pairs of primers, multiplex PCR to amplify 4 target sequences containing seven common genotypes of TNF gene G8226A, G-308A; A1031G, G863T and G857A; G8146C, C5478T, the product sizes are 420bp, 464bp, 165bp respectively , 282bp, and the primer sequences (SEQ ID NO.43-50...

Embodiment 3

[0095] The liquid phase chip of embodiment 3 different ASPE primers is to the detection of TNF gene SNP site

[0096] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0097] Taking the TNF gene G8226A, A1031G, G857A and G8146C 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 G8226A, A1031G, G857A and G8146C, respectively, and the ASPE primer 5 The Tag sequence at the 'end is selected from SEQIDNO.1-SEQIDNO.14, and correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQIDNO.29-SEQIDNO.42. The specific design is shown in the following table (Table 8). 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.

[0098] Table 8 Design...

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Abstract

The invention discloses a TNF gene mutation detection liquid chip, and specific primers. The TNF gene mutation detection liquid chip mainly comprises: ASPE primers, wherein each ASPE primer is composed of 5'-terminal tag sequence, and 3'-terminal specific primer sequence targeting target gene mutation sites, and the specific primer sequence comprises SEQ ID No.15 and SEQ ID No.16 targeting G8226A site, SEQ ID No.17 and SEQ ID No.18 targeting G-308A site, SEQ ID No.19 and SEQ ID No.20 targeting A1031G site, SEQ ID No.21 and SEQ ID No.22 targeting G863T site, SEQ ID No.23 and SEQ ID No.24 targeting G857A site, SEQ ID No.25 and SEQ ID No.26 targeting G8146C site, and / or SEQ ID No.27 and SEQ ID No.28 targeting C5478T site; microballoons coated with different anti-tag sequences; and amplification primers. Self-agreement ratio of detection results of the liquid chip with detection results of sequencing is as high as 100%; and parallel detection of wild types and mutant types of a plurality of mutation sites is 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 TNF gene mutation detection and a liquid phase chip. Background technique [0002] Tumor necrosis factor (tumor necrosis factor, TNF) is located on the short arm of chromosome 6 6p21.3 and is a member of the tumor necrosis factor superfamily. Tumor necrosis factor is a cytokine mainly produced by macrophages. Its most obvious activity feature is that it can specifically kill tumor cells in vivo or in vitro, and has no obvious toxic effect on normal tissue cells. The lymphotoxin (Lymphotoxin, LT) produced by lymphocytes has high homology with tumor necrosis factor in DNA and amino acid sequence, especially the function is very similar, so the tumor necrosis factor produced by macrophages is named tumor necrosis factor α (TNF-α), the lymphotoxin produced by lymphocytes is named tumor necrosis factor β (TNFβ). As a cytok...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6827C12Q1/686C12Q2563/149C12Q2531/113
Inventor 刘丽许昌有
Owner SUREXAM BIO TECH