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
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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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