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Probe, primer and kit for detecting gene polymorphism of catechol-O-methyltransferase (COMT)

A technology of methyltransferase and gene polymorphism, applied in the field of molecular biology, can solve the problems of high cost, unsuitable detection, time-consuming and laborious, etc., and achieve the effect of simple operation, rapid screening and high sensitivity

Inactive Publication Date: 2021-02-02
上海利康精准医疗技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the commonly used mutation detection method is direct DNA sequencing method. PCR products can be directly analyzed by DNA sequence, which can clarify the mutation site, but it has the disadvantages of 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 gene polymorphism of catechol-O-methyltransferase (COMT)
  • Probe, primer and kit for detecting gene polymorphism of catechol-O-methyltransferase (COMT)
  • Probe, primer and kit for detecting gene polymorphism of catechol-O-methyltransferase (COMT)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1: probe and primer design

[0028] The present invention designs probe and primer sequences for the COMT Val158Met SNP site. 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.

[0029] Design probes and primers to realize that when the template does not exist at the annealing temperature, the probe is in a stem-loop state, including the loop sequence and the reverse complemen...

Embodiment 2

[0036] Embodiment 2: Detect different genotype standard products

[0037] 1. Use the plasmid COMT Val158Met to construct and prepare wild-type standard plasmids and mutant standard plasmids containing the rs4680 site of the target gene (the source of the plasmid and the synthesis of the plasmid containing the target gene were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The accuracy of the sequence was determined by sanger sequencing, the genotype of the wild-type standard plasmid rs4680 was GG; the genotype of the mutant standard plasmid rs4680 was AA. The standard plasmid DNA concentration was standardized to 10ng / ul.

[0038] 2. Using the probes and primers in Example 1.

[0039] 3. PCR reaction system:

[0040] 1) Add 7.5ul of PCR Mix, 0.5uM of primer 1 solution and 0.5uM of primer 2 solution, and 0.1uM of probe into each PCR reaction well, and then add the wild-type standard plasmid into 3 different PCR reaction wells respectively 2ul each of DNA, mutant st...

Embodiment 3

[0043] Embodiment 3: detection method performance analysis experiment

[0044] 1. Precision experiment

[0045] Take a copy of wild-type standard plasmid DNA and mixed-type DNA (the wild-type standard plasmid and mutant standard plasmid are mixed at a ratio of 1:1), and test 3 times a day for 5 consecutive days. The method in Example 2, the results suggest that the repeatability of the fluorescent PCR amplification reaction is good (the coincidence rate is greater than 95%, and the coefficient of variation CV of the Ct value of the test result is less than 5%).

[0046] 2. Conformity rate experiment

[0047] Select 20 cases of DNA samples from healthy volunteers in Shanghai, apply the method of Example 2 to detect the rs4680 locus, and apply the Sanger sequencing method to verify at the same time, compare the consistency of the detection results of the two methods, and the results show that the method of Example 2 can be classified The coincidence rate of the result and the ...

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Abstract

The invention provides a probe for detecting the polymorphism of a catechol-O-methyltransferase (COMT). The sequence of the probe is shown as SEQ ID NO.: 1, or the two ends of the sequence of SEQ ID NO.: 1 are modified by groups. The invention also provides a primer for detecting the gene polymorphism of the COMT. The sequences of the primer are shown as SEQ ID NO.: 2 (primer 1) and SEQ ID NO.: 3(primer 2). When used for detecting the gene polymorphism of the COMT, the probe and the primer for detecting the gene polymorphism of the COMT have the advantages of high sensitivity, good specificity, rapidness, high-throughput detection and the like.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to probes, primers and kits for detecting polymorphisms of catechol-oxygen-methyltransferase genes. Background technique [0002] The catechol-oxygen-methyltransferase encoded by the COMT gene is the main degrading enzyme of catecholamines, which play an important role in the signal transduction between sympathetic ganglion cells and effectors. The most common polymorphism of this gene is Val158Met. Molecular biology studies have shown that the 158th amino acid of the COMT gene is converted from Val to Met, and the activity of the encoded catechol-oxygen-methyltransferase will be reduced by about 3 ~4 times. [0003] The COMT gene is significantly correlated with children's reading comprehension ability, language expression ability, concentration, logical thinking ability, musical ability, dance talent and other traits, and its polymorphism can be used to evaluate children's innate...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12N15/11C12Q1/6851
CPCC12Q1/6888C12Q1/6851C12Q2600/156
Inventor 王校陆婧毛丹丹张奕
Owner 上海利康精准医疗技术有限公司
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