CYP1A1 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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  • CYP1A1 gene polymorphism detection specific primers and liquid chip
  • CYP1A1 gene polymorphism detection specific primers and liquid chip
  • CYP1A1 gene polymorphism detection specific primers and liquid chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 CYP1A1 gene polymorphism detection liquid chip mainly includes:

[0025] 1. ASPE Primers

[0026] Specific primer sequences were designed for wild-type and mutant types of eight common genotypes of CYP1A1 gene T109C, T97C, 133-134insT, T201A, C240A, A242G, C248A and G135T. ASPE primers consist of "Tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0027] Table 1 ASPE primer sequence of CYP1A1 gene (Tag sequence + specific primer sequence)

[0028]

[0029]

[0030] Each ASPE primer includes two parts, the 5' end is a specific tag sequence for the ami-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.

[0031] 2. Microsphere...

Embodiment 2

[0042] Example 2 Detection of samples using the CYP1A1 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] 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] Four pairs of primers were designed, and multiplex PCR amplified four target sequences containing eight common genotypes T109C, T97C, 133-134insT, T201A, C240A, A242G, C248A and G135T of the CYP1A1 gene in one step. Among them, 133-134insT, T201A, C240A, A2...

Embodiment 3

[0099] Example 3 Detection of CYP1A1 gene polymorphism site by liquid chip with different ASPE primers

[0100] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0101] Taking the CYP1A1 gene T201A and A242G 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 T201A and A242G, and the Tag sequence at the 5' end of the ASPE primer was selected from SEQ ID NO.1-SEQ ID NO.16, correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.33-SEQ ID NO.48. 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.

[0102] Table 8 Design of liquid phase chip preparation

[0103]...

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PUM

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Abstract

The invention discloses CYP1A1 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.17 and SEQ ID NO.18 aiming at T109C site; SEQ ID NO.19 and SEQ ID NO.20 aiming at T97C site; SEQ ID NO.21 and SEQ ID NO.22 aiming at 133-134insT site; SEQ ID NO.23 and SEQ ID NO.24 aiming at T201A site; SEQ ID NO.25 and SEQ ID NO.26 aiming at C240A site; SEQ ID NO.27 and SEQ ID NO.28 aiming at A242G site; SEQ ID NO.29 and SEQ ID NO.30 aiming at C248A site; and / or SEQ ID NO.31 and SEQ ID NO.32 aiming at G135T 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 CYP1A1 gene polymorphism detection specific primer and a liquid phase chip. Background technique [0002] P450 (Cytochromes P450) is the most important metabolic enzyme in the first phase of biotransformation of exogenous chemical substances in the body. Pre-carcinogens enter the body and are catalyzed by CYP450 (including epoxidation, decyclylation, oxidation, reduction, hydrolysis, etc.) Various types of chemical processes), transform into electrophilic compounds to attack intracellular biomacromolecules, and initiate carcinogenic or mutagenic processes. Cytochrome CYP450 is a group of structurally and functionally related isoenzymes encoded by superfamily genes, belonging to hemoglobin enzymes, and its subfamily CYP1A1 is closely related to smoking-induced lung cancer. [0003] CYP1A1 is an important member of the CYP450 family, locate...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11G01N21/64
Inventor 许嘉森何嘉英
Owner SUREXAM BIO TECH
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