Probe and method for detecting polymorphism of TREM2 gene

A gene polymorphism and probe technology, applied in biochemical equipment and methods, microbial measurement/testing, DNA/RNA fragments, etc., can solve the problems of high cost, unsuitable detection, time-consuming and labor-intensive, etc., and achieve high sensitivity , clear genotyping, and simple method operation

Inactive Publication Date: 2019-06-04
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 method for detecting TREM2 gene mutations is direct DNA sequencing. PCR products can be directly analyzed for DNA sequences, which can clarify the mutation site, but it is time-consuming, laborious, expensive, and not suitable for detection of a large number of samples.

Method used

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  • Probe and method for detecting polymorphism of TREM2 gene
  • Probe and method for detecting polymorphism of TREM2 gene
  • Probe and method for detecting polymorphism of TREM2 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] 1. Construct and prepare the wild-type standard plasmid and mutant standard plasmid containing the target gene locus. The accuracy of the sequence can be confirmed by enzyme digestion and sequencing. The genotype of the wild-type standard plasmid rs75932628 is GG; the genotype of the mutant standard plasmid rs75932628 is AA. The standard plasmid DNA concentration was normalized to 10ng / μL.

[0037] 2. Use the online software Primer 3 to design primers to strengthen the complementary sequence of the primer loop and the target DNA sequence. The sequence of the forward primer is: 5′-

[0038] AGGTGACACTATAGAATAAGTTGTGCGTGCTGACCA-3′;

[0039] The sequence of the reverse primer is: 5'-

[0040] GTACGACTCACTATAGGGAAGTCTTGCCCCTATGACTCCA-3′;

[0041] The sequence of the probe is 5′-FAM-

[0042] CGGCACCAGGCCTTGGGCCTCCCGTGCCG-BHQ-3'.

[0043] 3. Reaction system optimization:

[0044] 1) Probe volume: set probe volumes of 0.01 μL, 0.05 μL, 0.1 μL and 0.5 μL respectively, an...

Embodiment 2

[0051] 1. Using the silica gel adsorption method to extract the genomic DNA of the oral epithelial cells of a test subject, the concentration and purity of the DNA were detected by electrophoresis gel imaging, and the concentration of the sample to be tested was standardized to 10 ng / μL.

[0052] 2. The detection method is as follows: add 7.5 μL of PCR Mix, 0.3 μL of forward primer solution, 0.3 μL of reverse primer solution, and 0.1 μL of probe to the PCR reaction wells in sequence. Add 2 μL of DNA to each reaction well, and make up 15 μL with sterilized double distilled water; carry out the reaction on the fluorescent quantitative PCR detection system SLAN-96P, and the PCR reaction conditions are 95°C pre-denaturation for 5 minutes; 95°C denaturation for 30 seconds, 60°C annealing for 30 seconds , 72°C extension for 30 seconds, 45 cycles; 72°C extension for 10 minutes; 95°C denaturation for 1 minute, 40°C renaturation for 1 minute, real-time monitoring of fluorescence signals...

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Abstract

The invention discloses a method for detecting polymorphism of a TREM2 gene, wherein the method includes the following steps: determining a mutation site of the TREM2 gene; designing and synthesizinga probe and primers according to the mutation site; mixing the detected TREM2 target gene, the probe, the primers and enzymes, carrying out fluorescence quantitative PCR, and monitoring a fluorescencesignal; and calculating the Tm value displayed by hybrid peaks, and judging the type of the TREM2 gene according to the Tm value. The invention also discloses the probe for detecting the polymorphismof the TREM2 gene, and the probe has the nucleotide sequence including SEQ ID NO.3. The invention provides the probe and method for simple operation and rapid detection of the polymorphism of the TREM2 gene.

Description

technical field [0001] The invention relates to a probe and method for detecting TREM2 gene polymorphism. Background technique [0002] The TREM2 gene is located on chromosome 6, and its extracellular domain may be related to cell recognition, while the intracellular domain is often combined with DAP12 to participate in other downstream signaling pathways. In recent years, the overexpression of TREM2 gene has been found in Alzheimer's disease patients and animal models. In animal models, TREM2 gene expression has even been found to be positively correlated with the regional distribution of Aβ and the content of Aβ. Therefore, it is speculated that TREM2 may be related to the recognition and phagocytosis of Aβ. . The TREM2 gene encodes a type 2 myeloid cell triggering receptor, which has anti-inflammatory effects in the brain, and the decrease in TREM2 activity may lead to increased inflammatory responses and brain damage. Gene-wide analysis further demonstrated that the TR...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12Q1/6883C12N15/11
Inventor 张奕毛丹丹
Owner 上海利康精准医疗技术有限公司
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