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CYP1A2 gene polymorphism detection specific primers and liquid chip

A technology for gene polymorphism and detection solution, applied in the field of molecular biology, can solve problems such as inability to use and cannot meet practical applications, and achieve consistent detection results, avoid uncertain factors, and avoid cross-reaction effects.

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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  • CYP1A2 gene polymorphism detection specific primers and liquid chip
  • CYP1A2 gene polymorphism detection specific primers and liquid chip
  • CYP1A2 gene polymorphism detection specific primers and liquid chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 CYP1A2 gene polymorphism detection liquid chip mainly includes:

[0024] 1. ASPE Primers

[0025] Specific primer sequences were designed for wild-type and mutant types of seven common genotypes of CYP1A2 gene, G216A, C96A, C139T, G100A, A141T, C113T and G110A. 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 CYP1A2 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...

Embodiment 2

[0042] Example 2 Detection of samples using the CYP1A2 gene polymorphism detection liquid chip described in Example 1

[0043] The formula of described various solutions is as follows:

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

[0045]

[0046] 2×Tm hybridization buffer

[0047]

[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] Nine pairs of primers were designed, and multiplex PCR amplified seven target sequences containing seven common genotypes of CYP1A2 gene G216A, C96A, C139T, G100A, A141T, C113T and G110A in one step. The product sizes were 340bp, 227bp, 306bp, 258bp, 305bp, 309bp and 296b...

Embodiment 3

[0098] Example 3 Detection of CYP1A2 gene polymorphism site by liquid chip with different ASPE primers

[0099] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0100] Taking the CYP1A2 gene C139T and A141T site mutation detection liquid chip as an example, the specific primer sequence at the 3' end of the ASPE primer was designed for the wild type and mutant type of C139T and A141T, and the Tag sequence at the 5' end of the ASPE primer was selected from SEQ ID NO.1-SEQ ID NO.14, correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.29-SEQ ID NO.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.

[0101] Table 8 Design of liquid phase chip preparation

[0102]...

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Abstract

The invention discloses CYP1A2 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.15 and SEQ ID NO.16 aiming at G216A site; SEQ IDNO.17 and SEQ ID NO.18 aiming at C96A site; SEQ ID NO.19 and SEQ ID NO.20 aiming at C139T site; SEQ ID NO.21 and SEQ ID NO.22 aiming at G100A T site; SEQ ID NO.23 and SEQ ID NO.24 aiming at A141T site; SEQ ID NO.25 and SEQ ID NO.26 aiming at C113T site; and / or SEQ ID NO.27 and SEQ ID NO.28 aiming at G110A 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 CYP1A2 gene polymorphism detection specific primer and a liquid phase chip. Background technique [0002] P450 enzyme belongs to monooxygenase (monooxygenase), also known as multifunctional oxidase (mixed function oxidase), hydroxylase (hydroxylase), named for its reduced state absorption peak at 450nm. It is mainly distributed in the endoplasmic reticulum, but also exists in the membranes of plasma membranes, mitochondria, Golgi bodies, peroxisomes, nuclear membranes and other organelles. It has a detoxification effect and can usually metabolize fat-soluble toxic substances into water-soluble substances. Toxic substances are excreted from the body. [0003] The cytochrome P450 enzyme system is a superfamily consisting of enzyme proteins encoded by homologous genes. There are 57 CYP genes in the human genome, which encode corresponding CY...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11G01N21/64
Inventor 许嘉森吴诗扬
Owner SUREXAM BIO TECH
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