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Probe, primer and kit for detecting AK4 gene polymorphism

A gene polymorphism and kit technology, applied in the field of molecular biology, can solve the problems of inapplicable detection of a large number of samples, time-consuming and labor-intensive, high cost, etc.

Inactive Publication Date: 2021-04-13
上海利康精准医疗技术有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the commonly used detection method of gene polymorphism is DNA direct sequencing method, PCR products can be directly analyzed by DNA sequence, and mutation sites can be identified, but there are disadvantages such as time-consuming, laborious, expensive, and not suitable for detection of a large number of samples.

Method used

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  • Probe, primer and kit for detecting AK4 gene polymorphism
  • Probe, primer and kit for detecting AK4 gene polymorphism
  • Probe, primer and kit for detecting AK4 gene polymorphism

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Embodiment 1: probe and primer design

[0044] The present invention designs probes and primer sequences for three SNP sites of AK4 gene. The specific principle is to use the conformational change of the fluorescent probe and the target sequence after hybridization to release the fluorescent dye, and judge the genotyping results according to the peak diagrams and Tm values ​​at different temperatures after hybridization. In the absence of target DNA, the fluorophore and quencher can be stably combined together, and no fluorescent signal can be detected; when there is target DNA, the structure of the fluorescently labeled probe is destroyed, and the fluorophore and quencher The extinguishing groups are separated from each other, and the fluorescent signal can be detected.

[0045] Design the probe and primers so that the probe is in a stem-loop state in the absence of the template at the annealing temperature. Taking AK4rs17853973 as an example, it includes the loop se...

Embodiment 2

[0062] Embodiment 2: Detect different genotype standard products

[0063] 1. Use plasmids to construct and prepare wild-type standard plasmids and mutant standard plasmids containing the target gene AK4 rs17853973, rs12074520, and rs4915685 sites (the source of the plasmid and the synthesis of the plasmid containing the target gene are provided by Sangon Bioengineering (Shanghai) Co., Ltd. Synthesized by Sanger sequencing to determine the accuracy of the sequence, the wild-type standard plasmid rs17853973 genotype is TT, rs12074520 genotype is CC, rs4915685 genotype is GG; the mutant standard plasmid rs17853973 genotype is AA, rs12074520 genotype It is AA, and the genotype of rs4915685 is AA. The standard plasmid DNA concentration is normalized to 10ng / ul.

[0064] 2. Using the probes and primers in Example 1.

[0065] 3. PCR reaction system:

[0066] 1) Add 7.5ul of PCR Mix, 0.5uM of 3 sets of forward primer solutions and 0.5uM of each of 3 sets of reverse primer solutions ...

Embodiment 3

[0069] Example 3: Double-blind experimental investigation using the AK4 gene polymorphism detection kit

[0070] 1. The genomic DNA of oral epithelial cells of 100 cases of healthy volunteers in Shanghai was extracted by silica gel adsorption method, and the concentration and purity of DNA were detected by electrophoresis gel imaging. The DNA concentration of the samples to be tested was standardized to 10ng / ul.

[0071] 2. The detection method is as follows: Add 7.5ul of PCR Mix, 0.5uM of each forward primer solution, 0.5uM of each reverse primer solution, and 0.1uM of each probe to each PCR reaction well, and carry out a weak positive control (rs17853973 gene rs12074520 genotype is AT, rs12074520 genotype is AC, rs4915685 genotype is AG), negative control (rs17853973 genotype is TT, rs12074520 genotype is CC, rs4915685 genotype is GG) and samples to be tested, add DNA 2ul, make up 15ul with sterilized double distilled water; react on a fluorescent quantitative PCR detector, ...

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Abstract

The invention discloses a probe, primer and kit for detecting AK4 gene polymorphism. The sequence of the probe is shown as SEQ ID NO.: 1, SEQ ID NO.: 4 or SEQ ID NO.: 7, or the two ends of each of the sequences of SEQ ID NO.: 1, SEQ ID NO.: 4 and SEQ ID NO.: 7 are subjected to group modification. The probe, primer and kit for detecting the AK4 gene polymorphism have the advantages of high sensitivity, good specificity, rapidness, high-throughput detection and the like during detection of AK4 gene polymorphism.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to probes, primers and kits for detecting AK4 gene polymorphisms. Background technique [0002] Adenylate kinase (AK) family isoenzymes widely exist in organisms, and are machines for intracellular nucleotide synthesis, involved in intracellular energy and nucleotide metabolism. They reversibly catalyze the transfer of phosphate groups between ATP and AMP, and release 2 molecules of ADP. The AK4 gene is located on 1p31, and the enzyme is mainly expressed in mitochondria-rich tissues such as brain, heart, kidney and liver. AK4 can not only catalyze the phosphorylation of dAMP, CMP and dCMP with ATP or GTP as a phosphate donor, but also phosphorylate AMP with UTP as a phosphate donor. Studies have shown that AK4 can regulate the balance of adenosine, thereby affecting the survival and proliferation of cells. The expression of AK4 gene in human hepatoma cell line (HepG2) and human e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2531/113C12Q2563/107C12Q2545/114
Inventor 傅咏南沈勋章张奕毛丹丹
Owner 上海利康精准医疗技术有限公司
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