CDKN1B gene mutation detection specific primers and liquid chip

A detection solution and specific technology, applied in the field of molecular biology, can solve the problems of inappropriate clinical diagnostic chips, inability to use gene mutation detection, and expensive detection, so as to avoid uncertain factors, consistent detection results, and avoid crossover. effect of reaction

Active Publication Date: 2014-06-18
SUREXAM BIO TECH
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AI Technical Summary

Problems solved by technology

[0003] At present, CDKN1B gene mutation detection methods mainly include: PCR-RFLP technology, fluorescent quantitative PCR technology and Affymetrix chip technology. PCR-RFLP method is based on the change of restriction enzyme recognition site caused by gene mutation, such as site loss or Generate a new site, amplify a specific fragment by PCR, then digest the amplified product with a restriction endonuclease, and observe the size of the fragment by electrophoresis. Determine the genotype, but this method cannot be used for the detection of gene mutations that do not generate new restriction sites
Fluorescent quantitative PCR technology has the characteristics of high sensitivity, strong specificity, and high degree of automation, but it also has the disadvantages of easy sample contamination and high false positive rate, and can only detect one mutation type at a time
Although high-throughput means that Affymetrix chip technology is relatively mature, but this chip technology is not suitable for low-to-medium density clinical diagnostic chips, and it is difficult to expand the detection of SNPs or tagged SNPs related to many biological traits in the same reaction system
In addition, the Affymetrix chip is mainly strong on the expression profile chip, with many species, relatively weak on the SNP chip, and the detection price is expensive, which cannot meet the actual needs

Method used

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  • CDKN1B gene mutation detection specific primers and liquid chip
  • CDKN1B gene mutation detection specific primers and liquid chip
  • CDKN1B gene mutation detection specific primers and liquid chip

Examples

Experimental program
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Effect test

Embodiment 1

[0020] Embodiment 1 CDKN1B gene mutation detection liquid chip mainly includes:

[0021] 1. ASPE Primers

[0022] Specific primer sequences were designed for wild-type and mutant types of three common genotypes of CDKN1B gene, G-79A, G-838T and T326G. ASPE primers consist of "tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0023] Table 1 ASPE primer sequence of CDKN1B gene (tag sequence + specific primer sequence)

[0024]

[0025]

[0026] 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 / L Tris Buffer.

[0027] 2. Microspheres coated with anti-tag sequences ...

Embodiment 2

[0038] Example 2 Detection of samples using the CDKN1B 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] Three pairs of primers were designed, and multiplex PCR amplified three target sequences containing three common genotypes G-79A, G-838T and T326G of the CDKN1B gene in one step. The product sizes were 242bp, 380bp, and 283bp, respectively. .19-24) See Table 3 above.

[0051...

Embodiment 3

[0093] Example 3 Detection of CDKN1B gene SNP site by liquid chip with different ASPE primers

[0094] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0095] Taking the CDKN1B gene G-79A, G-838T and T326G 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 G-79A, G-838T and T326G, respectively, The Tag sequence at the 5' end of the ASPE primer is selected from SEQ ID NO.1-SEQ ID NO.6. Correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO. 13 - SEQ ID NO. 18. 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.

[0096] Table 7 Design of liquid phase chip p...

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Abstract

The invention discloses a CDKN1B gene mutation detection liquid chip, and specific primers. The CDKN1B 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.7 and SEQ ID No.8 targeting G-79A site, SEQ ID No.9 and SEQ ID No.10 targeting G-838T site, and / or SEQ ID No.11 and SEQ ID No.12 targeting T326G site; microballoons coated with 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 CDKN1B gene mutation detection specific primer and a liquid phase chip. Background technique [0002] The full name of the CDKN1B gene is cyclin dependent kinase inhibitor 1B (cyclin dependent kinase inhibitor 1B, CDKN1B), also known as p27, Kip1, located on the short arm of chromosome 12 12p13. important genes. The P27 protein encoded by the CDKN1B gene is a cyclin-dependent protein kinase inhibitor, and the encoded protein contains 198 amino acids with a relative molecular mass of about 27×10 3 , consisting of 2 exons and 2 introns. There is no structural zinc finger domain at the N-terminus of the P27 protein, and there are two separate nuclear localization signals at the C-terminus. p27 mainly binds to cyclin to exert an inhibitory effect on cyclin2CDK, and has multiple biological functions: (1) It can directly inhibit the biologica...

Claims

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Application Information

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