AGT1R (Angiotensin Type 1 Receptor) gene SNP (Single Nucleotide Polymorphism) detection liquid phase chip and specific primer

A detection solution and specific technology, applied in the field of molecular biology, can solve the problems of high false positive rate, easy contamination of samples, low sensitivity, etc., and achieve the effect of avoiding cross-reaction, strong scalability, and improved sensitivity.

CN102021236AInactive Publication Date: 2011-04-20SUREXAM BIO TECH
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Publication Date
2011-04-20
Estimated Expiration
Not applicable · inactive patent

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Abstract

The invention discloses an AGT1R (Angiotensin Type 1 Receptor) gene SNP (Single Nucleotide Polymorphism) detection specific primer and a liquid phase chip. The liquid phase chip mainly comprises an ASPE (Allele Specific Primer Extension) primer, a microsphere and an amplification primer, wherein the ASPE primer consists of tag sequences at an ends 5' and specific primer sequences specific to SNP sites at ends 3', the specific primer sequences are respectively selected from SEQ ID NO.5, SEQ ID NO.6, and / or SEQ ID NO.7 and SEQ ID NO.8, and the tag sequences are selected from SEQ ID NO.1-SEQ ID NO.4. The prepared AGT1R gene SNP detection liquid phase chip has excellent signal-noise ratio, and cross reaction does not exist between a designed probe and an anti-tag sequence basically. The ASPE primer designed by the invention has excellent specificity, and can accurately differentiate various kinds of mutant sites.
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Description

technical field

[0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to an AGT1R gene SNP detection liquid chip and specific primers. Background technique

[0002] Angiotensin II (angiotensin II, Ang II) is the main effector molecule in the renin-angiotensin-aldosterone system (rennin-angiotensinaldosterone system, RAAS) to regulate vasoconstriction and maintain blood volume, and the angiotensin II receptor (angiotensin II receptor, ATR) It mediates the physiological effects of AngII and is divided into two subtypes: type I angiotensin II receptor (AT1, AT1R) and type II angiotensin II receptor (AT2R). Among them, the main biological effect is AT1R, AT1R mainly regulates the activity of AngII, participates in the vasoconstriction and positive inotropic effect of AngII, including vasoconstriction, pressor response, renal tubular sodium ion transport and aldosterone secretion, and is a key link in the action of RA...

Examples

Embodiment 1A

[0019] Example 1 The AGT1R gene SNP detection liquid chip mainly includes:

[0020] 1. ASPE Primers

[0021] Specific primer sequences were designed for the wild type and mutant type of two common genotypes A1166C and A-153G of the AGT1R gene. ASPE primers consist of "Tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0022] The ASPE primer sequence (Tag sequence+specific primer sequence) of table 1AGT1R

[0023]

[0024] 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 / LTrisBuffer.

[0025] 2. Microspheres coated with anti-tag sequences

[0026] According to t...

Embodiment 2

[0036] Example 2: Detection of samples using the AGT1R gene detection liquid chip described in Example 1

[0037] The formula of described various solutions is as follows:

[0038] 50mM MES buffer (pH5.0) formula (250ml):

[0039] Reagent

[0040] MES(2[N-Morpholino]

[0041] 2×Tm hybridization buffer

[0042] Reagent

[0043] Store at 4°C after filtration.

[0044] ExoSAP-IT kit was purchased from US USB Company.

[0045] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0046] 1. Sample DNA extraction:

[0047] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0048] 2. PCR amplification of samples to be tested

[0049] Using Primer5.0 to design two pairs of primers, multiplex PCR amplified two target sequences containing the SNP mutation sites of two common genotypes A1166C and A-153G of the AGT1R gene in one step, and the product s...

Embodiment 3

[0108] The liquid phase chip of embodiment 3 different ASPE primers detects the SNP site of AGT1R gene

[0109] 1. Design of liquid phase chip preparation (selection of TAg sequence and Anti-Tag sequence)

[0110] Taking the AGT1R gene A1166C 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 A1166C, and the Tag sequence at the 5' end of the ASPE primer was selected from SEQ ID NO.1-SEQ ID NO .4. Correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.9-SEQ ID NO.12. 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.

[0111] Table 7 Design of liquid phase chip preparation

[0112]

[0113] 2. Samp...