Specific primes and liquid phase chip for AGT (angiotensinogen) and ACE (angiotensin-converting enzyme) gene polymorphism detection

A technology for gene polymorphism and detection solution, applied in the field of molecular biology, can solve the problems of inability to detect gene mutation, cannot meet practical application, prone to cross-reaction, etc., and achieves avoiding uncertain factors and good signal-to-noise ratio. , the detection effect is consistent

Active Publication Date: 2014-05-07
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Fluorescent quantitative PCR has the advantages of simple operation, quick results, and quantification. However, this technology has the disadvantages of easy sample contamination, cross-reaction, and high false positive rate; while PCR-RFLP method is based on restriction endonucleation caused by gene mutations. Changes in the enzyme recognition site, such as site loss or new site generation, a specific fragment is amplified by PCR, and then the amplified product is digested with a restriction endonuclease, and the size of the fragment is observed by electrophoresis. This method is used for Detection of gene mutations with altered restriction sites can directly determine the genotype, but 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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  • Specific primes and liquid phase chip for AGT (angiotensinogen) and ACE (angiotensin-converting enzyme) gene polymorphism detection
  • Specific primes and liquid phase chip for AGT (angiotensinogen) and ACE (angiotensin-converting enzyme) gene polymorphism detection
  • Specific primes and liquid phase chip for AGT (angiotensinogen) and ACE (angiotensin-converting enzyme) gene polymorphism detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 AGT and ACE gene polymorphism detection liquid chip mainly includes:

[0027] 1. ASPE Primers

[0028] Specific primer sequences were designed for wild-type and mutant types of T185C, A75G and C99T common mutation sites of AGT gene, and insertion / deletion (I / D) of common genotypes of ACE gene. ASPE primers consist of "Tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0029] Table 1 ASPE primer sequences of AGT and ACE genes (Tag sequence + specific primer sequence)

[0030]

[0031] 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.

...

Embodiment 2

[0044] Example 2 Detection of Samples Using AGT and ACE Gene Polymorphism Detection Liquid Chip

[0045] The formula of described various solutions is as follows:

[0046] 50mM MES buffer (pH5.0) formula (250ml):

[0047]

[0048] 2×Tm hybridization buffer

[0049] Reagent

source

Final concentration

Dosage per 250ml

1M Tris-HCl, pH8.0

SigmaT3038

0.2M

50ml

5M NaCl

Sigma S5150

0.4M

20ml

Triton X-100

Sigma T8787

0.16％

0.4ml

[0050] Store at 4°C after filtration.

[0051] ExoSAP-IT kit was purchased from US USB Company.

[0052] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0053] 1. Sample DNA extraction:

[0054] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0055] 2. PCR amplification of samples to be tested

[0056] Design four pairs of primers a...

Embodiment 3

[0112] Example 3 Detection of AGT and ACE gene polymorphism sites by liquid chip with different ASPE primers

[0113] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0114] Taking the AGT gene T185C site mutation and the ACE gene insertion / deletion 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, mutant type, and insertion / deletion type of T185C, and the ASPE The Tag sequence at the 5' end of the primer is selected from SEQ ID NO.1-SEQ ID NO.8. Correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.17- SEQ ID NO. 24. 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.

[0115...

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Abstract

The invention discloses specific primes and a liquid phase chip for AGT (angiotensinogen) and ACE (angiotensin-converting enzyme) gene polymorphism detection. The liquid phase chip mainly contains ASPE (allele specific primer extension) which consists of tag sequences at 5' end and specific primers at 3'-end relative to a target gene mutation, wherein the specific primers respectively are SEQ ID NO. 9 and SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14 regarding to relative to the AGT gene, and / or SEQ ID NO. 15 and SEQ ID NO. 16 relative to the ACE gene; microspheres coated with anti-tag sequences; and amplification primers. The conformation between the detection result of the AGT and ACE gene polymorphism detection liquid phase chip and the result of a sequencing method is up to 100%. The prepared liquid phrase has excellent signal-noise ratio and can avoid cross reaction and realize the parallel detection of multiple polymorphic sites.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a specific primer for detecting AGT and ACE gene polymorphism and a liquid phase chip. Background technique [0002] Renin-angiotensin-aldosterone (renin-angiotensin system, RAS) is a hormone system that plays an important role in the regulation of blood pressure, blood flow and internal environment. Among them, angiotensinogen and angiotensin converting enzyme It is a key component of the RAS system. [0003] Angiotensinogen (Angiotensinogen, AGT) is an α-2 globulin that is continuously synthesized by the liver and released into the blood circulation. The main function of AGT is to play an important role in regulating blood pressure as a substrate of renin. Angiotensin-Converting Enzyme (ACE) is also known as dipeptide carboxypeptidase. The main function of ACE is to convert angiotensin I into angiotensin II, which has a strong vasoconstri...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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