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Kit and method for designing primer for human ABCC2 gene polymorphism detection

A technology of gene polymorphism and primer design, applied in the fields of clinical laboratory science and molecular biology, to achieve low-cost results

Active Publication Date: 2019-04-05
CHINA JAPAN FRIENDSHIP HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The detection method of the gene polymorphism of the present invention not only has the advantages of quickness, simplicity, accuracy, high throughput and low cost, but also overcomes the defects of conventional HRM primer design

Method used

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  • Kit and method for designing primer for human ABCC2 gene polymorphism detection
  • Kit and method for designing primer for human ABCC2 gene polymorphism detection
  • Kit and method for designing primer for human ABCC2 gene polymorphism detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Example 1. Synthetic primers for human ABCC2 gene polymorphism detection

[0100] Using the primer 5.0 software, using the -24C>T, 1249G>A, and 3972C>T sites of the human ABCC2 gene as detection sites, the upstream and downstream primer sequences were designed according to the following design method, and the following 6 principles were met at the same time:

[0101] (1) Make the gene polymorphism site in the middle position, and design upstream and downstream primers with a gene sequence of a total length of 400 bp including the site;

[0102] (2) In the PCR reaction system, the final primer concentration is 1-3 μM;

[0103] (3) The total length of each product is ≤160bp;

[0104] (4) The annealing temperature Tm is 58-62°C;

[0105] (5) HPLC purification is carried out to the synthetic primer sequence;

[0106] (6) Mg in the system 2+ The final concentration of 2.0 ~ 2.5mM.

[0107] The sequence sources of the -24C>T, 1249G>A, 3972C>T sites of the human ABCC2 gen...

Embodiment 2

[0113] Example 2. The positive control sequence for the detection of polymorphisms in the synthetic human ABCC2 gene

[0114] Design the positive control sequence for human ABCC2 gene polymorphism detection. The positive sequence is directly synthesized using a DNA sequence synthesizer according to the wild-type and mutant gene sequences. Entrust Beijing Qingke Xinye Biotechnology Co., Ltd. to synthesize the positive control sequence, see Table 2

[0115] Table 2: Positive control for polymorphism detection of human ABCC2 gene, underlined bases are mutation sites

[0116]

[0117]

Embodiment 3

[0118] Example 3. A kit for detecting the polymorphism of the ABCC2 gene

[0119] Utilize the primer design method of the present invention, utilize HRM technology, prepare the test kit that detects ABCC2 gene polymorphism

[0120] 1. The composition of the kit

[0121] 1. Reagents for extracting ABCC2 gene DNA from the patient's whole blood, including the following reagents packaged separately:

[0122] 1) Lysis solution: solution containing guanidine isothiocyanate (Tiangen DP318-02 Blood Genomic DNA Extraction Kit);

[0123] 2) Protein removal solution: a solution containing isopropanol (Tiangen DP318-02 Blood Genomic DNA Extraction Kit); 3) Proteinase K, 40unit / mg, 100mg (Solarbio, purchased from Beijing Soleibao Technology Co., Ltd.);

[0124] 4) Rinse solution: 200 mL of ethanol solution with a concentration of 75% by volume;

[0125] 5) Eluent: deionized water 200mL;

[0126] 6) Adsorption column: the adsorption column CB3 in the DP318-02 Blood Genomic DNA Extractio...

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Abstract

The invention discloses a kit and a method for designing a primer for human ABCC2 gene polymorphism detection and belongs to the technical field of clinical laboratory medicine and molecular biology.The method for designing the primer for human ABCC2 gene polymorphism detection meets the following conditions: (1) a gene polymorphism site is located on a middle position and upstream and downstreamprimers are designed on the basis of a gene sequence in length of 400bp including the site; (2) final concentration of primers is 1-3uM in a PCR reaction system; (3) total length of each product is less than or equal to 160bp; (4) annealing temperature Tm is at 58-62 DEG C; (5) HPLC purification is performed on the compound primer sequence; (6) final concentration of Mg2+ in the system is 2.0-2.5mM. The invention has the advantages: the primer design method is capable of avoiding the interference of a high resolution melting curve technique for detecting gene polymorphism; ABCC2 gene kit amplifies while directly splits, with high flux, high speed, high accuracy and high specificity; the ABCC2 gene kit can be used for finding a new target with individual difference; a basis can be suppliedfor clinical individualized medication decisions.

Description

technical field [0001] The present invention relates to a primer design method and a kit for detecting human ABCC2 gene polymorphisms, especially to a primer design method for detecting human ABCC2 gene polymorphisms using HRM technology and the human ABCC2 gene polymorphisms established by this method The detection kit belongs to the technical fields of clinical laboratory science and molecular biology. Background technique [0002] The era of precision medicine has arrived. Gene polymorphisms have a great influence on the drug responsiveness of different patients, and it is found that more and more drugs need to be carried out under the guidance of genes, so genetic testing is of great significance to individualized treatment. [0003] ABCC2 is the coding gene of multidrug resistance-related protein 2 (MRP2), which is located in the chromosome 10q24 region. The length of the gene is about 45kbp, including 32 exons. The MRP2 membrane protein in hepatocytes participates in...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/106C12Q2600/156C12Q2531/113C12Q2527/137C12Q2527/107
Inventor 陈利达马亮曹永彤李金明刘倩杨辉刘媛媛芦宏凯姜蕾王璐璐
Owner CHINA JAPAN FRIENDSHIP HOSPITAL
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