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CYP17A1 (Cytochrome P450) genetic polymorphism detection specific primer and liquid-phase chip

A technology for gene polymorphism and detection solution, applied in the field of molecular biology, can solve the problems of high false positive rate, low sensitivity, easy contamination of samples, etc., and achieve the effects of good detection specificity, simple steps and consistent detection effect.

Active Publication Date: 2012-07-04
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the products for detecting CYP17A1 are generally RT-PCR, fluorescent quantitative PCR, traditional solid-phase chip technology, direct sequencing, PCR-RFLP, etc. based on PCR technology. This technology has the disadvantages of low sensitivity, easy sample contamination, and high false positive rate. , at the same time, due to the limitation of the detection flux, it cannot meet the needs of practical applications

Method used

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  • CYP17A1 (Cytochrome P450) genetic polymorphism detection specific primer and liquid-phase chip
  • CYP17A1 (Cytochrome P450) genetic polymorphism detection specific primer and liquid-phase chip
  • CYP17A1 (Cytochrome P450) genetic polymorphism detection specific primer and liquid-phase chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 CYP17A1 gene polymorphism detection liquid chip mainly includes:

[0024] 1. ASPE Primers

[0025] Specific primer sequences were designed for wild type and mutant type of three common genotypes A221G, C193T and T81C of CYP17A1 gene. 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 CYP17A1 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 anti-tag sequences ...

Embodiment 2

[0042] Example 2 Detection of samples using CYP17A1 gene detection liquid chip

[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] Design three pairs of primers, multiplex PCR to amplify three target sequences of three common genotypes A221G, C193T and T81C containing CYP17A1 gene in one step, the product sizes are 322bp, 358bp and 440bp respectively, the primer sequences

[0055] (SEQ ID NO.19-24) are shown in Table 3 abov...

Embodiment 3

[0113] Example 3 Detection of CYP17A1 gene polymorphism by liquid chip with different ASPE primers

[0114] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0115] Taking the A221G site mutation detection liquid chip of the CYP17A1 gene 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 A221G, and the Tag sequence at the 5' end of the ASPE primer was selected from SEQ ID NO.1 -SEQ ID NO.6, correspondingly, the anti-tag sequence coated on the microspheres 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.

[0116] Table 7 Design of liquid phase chip preparation

[0117]

[0118] 2....

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Abstract

The invention discloses a CYP17A1 (Cytochrome P450) genetic polymorphism detection specific primer and liquid-phase chip. The liquid-phase chip mainly comprises an ASPE (Allele Specific Primer Extension) primer, microspheres and an amplification primer, wherein the ASPE primer consists of a tag sequence at 5' end and specific primers aiming at target gene mutation at 3' end, wherein the specific primers are SEQ ID NO. 7 and SEQ ID NO. 8 aiming at an A221G site, SEQ ID NO. 9 and SEQ ID NO. 10 aiming at C193T, and / or SEQ ID NO. 11 and SEQ ID NO. 12 aiming at T81C; and the microspheres are coated by anti-tag sequence. The matching ratio of the detection result of the CYP17A1 genetic polymorphism detection liquid-phase chip provided by the invention and that of a sequencing method reaches up to 100 percent. The prepared liquid-phase chip has excellent signal-noise ratio; and cross reaction can be avoided and parallel detection of a plurality of polymorphic sites can be realized through selection of a tag label sequence and an anti-tag label sequence and combination of the tag label sequence and a specific ASPE primer.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a CYP17A1 gene polymorphism detection specific primer and a liquid phase chip. Background technique [0002] Cytochrome oxidase P450 (CYP450) is a class of enzymes involved in the metabolism of endogenous and exogenous compounds. It mainly exists in the endoplasmic reticulum of organisms. Many monooxygenases, CYP450 enzymes have many isoenzymes, among them, cytochrome CYP17 enzyme plays an important role in the synthesis of adrenal and gonadal steroid hormones, and it mainly exerts the functions of two enzymes, namely 17α- Hydroxylase and 17,20-lyase, 17α-hydroxylase (17α-hydroxylase), also known as 17,20 carbon chain lyase, catalyzes the conversion of pregnenolone and progesterone into 17α-hydroxypregnenolone and 17α-hydroxylase Pregnancy (17αP), 17,20-lyase cleaves the 17,20 carbon chain to form the precursor of estrogen, namely dehydroep...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 许嘉森秦会娟郭婧刘志明
Owner SUREXAM BIO TECH
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