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CYP2B6 gene polymorphism detection specific primers and liquid chip

A gene polymorphism and detection solution 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 effects of avoiding uncertain factors, consistent detection results, and simple steps

Inactive Publication Date: 2013-02-06
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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  • CYP2B6 gene polymorphism detection specific primers and liquid chip
  • CYP2B6 gene polymorphism detection specific primers and liquid chip
  • CYP2B6 gene polymorphism detection specific primers and liquid chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 CYP2B6 gene polymorphism detection liquid chip mainly includes:

[0024] 1. ASPE Primers

[0025] Specific primer sequences were designed for wild-type and mutant types of six common genotypes of CYP2B6 gene, C123T, A101G, G118T, A77G, T139C and C288T. ASPE primers consist of "Tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0026] ASPE primer sequence (Tag sequence+specific primer sequence) of table 1 CYP2B6 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 an...

Embodiment 2

[0043] Example 2 Using the CYP2B6 gene polymorphism detection liquid chip described in Example 1 to detect samples The formulations of the various solutions are as follows:

[0044] 50mM MES buffer (pH5.0) formula (250ml):

[0045]

[0046] 2×Tm hybridization buffer

[0047] Reagent

[0048] Store at 4°C after filtration.

[0049] ExoSAP-IT kit was purchased from US USB Company.

[0050] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0051] 1. Sample DNA extraction:

[0052] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0053] 2. PCR amplification of samples to be tested

[0054] Five pairs of primers were designed, and multiplex PCR amplified five target sequences containing six common genotypes C123T, A101G, G118T, A77G, T139C and C288T of CYP2B6 gene in one step. They are 356bp, 235bp, 246bp, 275bp and 384bp respectively, and the primer seque...

Embodiment 3

[0100] Example 3 Detection of CYP2B6 gene polymorphism site by liquid chip with different ASPE primers

[0101] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0102] Taking the CYP2B6 gene A101G and T139C 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 A101G and T139C, and the Tag sequence at the 5' end of the ASPE primer was selected from SEQ ID NO.1-SEQ ID NO.12, correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.25-SEQ ID NO.36. 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.

[0103] Table 8 Design of liquid phase chip preparation

[0104]...

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Abstract

The invention discloses CYP2B6 gene detection specific primers and a liquid chip. The liquid chip mainly comprises: various ASPE primers composed of 5' terminal tag sequence and 3' terminal specific primer sequences aiming at target gene mutation site, microspheres coated with anti-tag sequence, and amplification primers. The specific primer sequences comprise: SEQ ID NO.13 and SEQ ID NO.14 aiming at C123T site; SEQ ID NO.15 and SEQ ID NO.16 aiming at A101G site; SEQ ID NO.17 and SEQ ID NO.18 aiming at G118T site; SEQ ID NO.19 and SEQ ID NO.20 aiming at A77G site; SEQ ID NO.21 and SEQ IDNO.22 aiming at T139C site; and / or SEQ ID NO.23 and SEQ ID NO.24 aiming at C288T site. The matching rate of the detection result obtained by using the liquid chip provided by the invention and a result obtained by using a sequencing method is 100%. Therefore, wild-type and mutant-type parallel detections of a plurality of mutation sites are realized.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a CYP2B6 gene polymorphism detection specific primer and a liquid phase chip. Background technique [0002] CYP2B6 is an important drug-metabolizing enzyme in the CYP family. It is widely distributed in human liver, kidney, lung, small intestine, endometrium, bronchoalveolar macrophages, peripheral blood lymphocytes and brain, etc., and participates in various endogenous and Synthesis and metabolism of xenobiotics. The CYP2B6 gene is located at 19q12-13.2, with a total length of 28kb, separated by 8 introns and 9 exons, and the encoded protein consists of 491 amino acids with a relative molecular mass of 58268. Studies have shown that the expression level of CYP2B6 protein varies not only between races, but also between different genders, with individual differences of more than 250 times, and even some people's expression cannot be detecte...

Claims

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Application Information

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