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ABCG2 gene 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 problems such as insufficiency, and achieve the effects of consistent detection effect, low cross-reaction rate 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

[0005] At present, there are few methods for detecting and analyzing ABCG2 gene polymorphisms. The only common detection method is direct sequencing, which is the gold standard method of genetic detection. However, due to the limitation of detection throughput, only It can detect a mutation type, which cannot meet the needs of practical applications

Method used

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  • ABCG2 gene polymorphism detection specific primer and liquid phase chip
  • ABCG2 gene polymorphism detection specific primer and liquid phase chip
  • ABCG2 gene polymorphism detection specific primer and liquid phase chip

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Example 1 ABCG2 gene polymorphism detection liquid chip mainly includes:

[0024] 1. ASPE Primers

[0025] Specific primer sequences were designed for the wild-type and mutant types of the three common genotypes C184T, C229A and C145T of the ABCG2 gene. ASPE primers consist of "Tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0026] Table 1 ASPE primer sequence (Tag sequence + specific primer sequence) of ABCG2 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 s...

Embodiment 2

[0041] Example 2 Detection of samples using the ABCG2 gene polymorphism detection liquid chip described in Example 1

[0042] The formula of described various solutions is as follows:

[0043] 50mM MES buffer (pH5.0) formula (250ml):

[0044]

[0045] 2×Tm hybridization buffer

[0046] Reagent

[0047] Store at 4°C after filtration.

[0048] ExoSAP-IT kit was purchased from US USB Company.

[0049] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0050] 1. Sample DNA extraction:

[0051] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0052] 2. PCR amplification of samples to be tested

[0053] Two pairs of primers were designed, and multiplex PCR amplified two target sequences containing three common genotypes C184T, C229A and C145T of the ABCG2 gene in one step. Among them, C184T and C229A were located in the same amplification product, and the pr...

Embodiment 3

[0096] Example 3 Detection of ABCG2 gene SNP site by liquid chip with different ASPE primers

[0097] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0098]Taking the ABCG2 gene C184T 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 C184T, 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 microsphere 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.

[0099] Table 7 Design of liquid phase chip preparation

[0100]

[0101] 2. Sample testing ...

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Abstract

The invention discloses an ABCG2 gene detection specific primer and a liquid phase chip. The liquid phase chip main comprises each ASPE primer, a microsphere coated by anti-tag sequence and an amplification primer, wherein the ASPE primer comprises 5' end tap sequence and 3' end specific primer sequence aiming at gene mutation site; and the specific primer sequence comprises SEQ ID NO.7 and SEQ ID NO.8 aiming at the C184T site, SEQ ID NO.9 and SEQ ID NO.10 aiming at the C229A site, and / or SEQ ID NO.11 and SEQ ID NO.12 aiming at the C145T site. The consistency between the detection result of the liquid phase chip and that of a sequencing method reaches 100 percent, so that multi-mutational-site wild type and mutant type parallel detection is realized.

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 ABCG2 gene polymorphism and a liquid phase chip. Background technique [0002] ABCG2 is the second member of subfamily G of ABC transporter superfamily (ATP-binding cassette transporter superfamily, ABC transporter superfamily). ABCG2 is located in the cell membrane and is widely distributed in normal tissues, mainly in tissues with secretory and excretory functions such as placental syncytiotrophoblast, small intestine and colon epithelium, liver canalicular membrane, bile canalicular membrane, breast lobules and vascular endothelial cells. The human ABCG 2 gene is located at 4q22-23, encoding 655 amino acid residues. The full length of ABCG 2 is 66kb, consisting of 16 exons and 15 introns, and the exons range from 60 to 332bp. Currently, more than 40 SNPs have been found in the ABCG2 gene, among which the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 许嘉森陈家欣
Owner SUREXAM BIO TECH
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