Primer for detecting mutation of EGF (Epidermal Growth Factor) gene
A kit and gene technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problem of simultaneous determination of multiple mutations of EGF gene, etc.
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Embodiment 1
[0079] Example 1: Design of Specific Amplification Primers
[0080] Design specific amplification primers according to the reference sequence of the EGF normal gene in the NCBI database, and the specific amplification primers should meet the following conditions:
[0081] (1) The product cannot form a secondary structure, the bases should be randomly distributed, and the primer itself cannot have 4 consecutive bases complementary;
[0082] (2) The annealing temperature difference between the forward and reverse primers of all PCR fragments should not exceed 5°C, and the annealing temperature of each product should be between 48°C and 58°C to ensure the specificity and stability of amplification;
[0083] (3) The forward amplification primer of the PCR amplification primer is the primer used for sequencing amplification, and the length is between 18-22bp.
[0084] The specific amplification primers designed in the present invention are shown in Table 1.
Embodiment 2
[0085] Embodiment 2: sample DNA extraction
[0086] The positive sample used in the present invention was taken from a subject in Shandong, who was diagnosed as hypomagnesemia type 4 by the hospital, and his blood sample was taken with the consent of the subject.
[0087] GentraPuregene blood DNA extraction kit (Qiagen, Germany) was used to extract blood genomic DNA according to the instructions, and the specific steps were as follows:
[0088] (1) Take 900 μL of erythrocyte lysate and put it into a 1.5 mL EP tube;
[0089] (2) Add 300 μL of whole blood to the above EP tube, mix upside down, and incubate at room temperature for 10 minutes to lyse red blood cells. Note that during the incubation period, mix upside down at least once;
[0090] (3) Centrifuge at 13200×g for 20 s at room temperature, and remove most of the supernatant with a tip (note that about 20 μL to 30 μL of supernatant is left in the tube);
[0091] (4) Vortex the upper tube to suspend the cells in the res...
Embodiment 3
[0102] Embodiment 3: PCR amplification
[0103] Using the specific amplification primers designed in Example 1, the sample DNA was used as a template for PCR amplification. The reaction system for PCR amplification was 50 μL, and the specific composition was: 3 μL of DNA template, each primer (forward and reverse) 2 μL, Taq enzyme 0.4 μL, dNTP 4 μL, 10×PCR buffer 5 μL, double distilled water to make up to a total volume of 50 μL.
[0104] The PCR amplification conditions are: pre-denaturation at 94°C for 3min, denaturation at 94°C for 35s, annealing for 35s, annealing temperature for specific amplification primers at 49°C-57°C, extension at 72°C for 50s, a total of 30 cycles; final extension at 72°C for 5min .
[0105] The annealing temperature of the specific amplification primers was investigated and optimized, and the optimized annealing temperatures of each specific amplification primer are shown in Table 1.
[0106] Table 1. Specific amplification primers and annealing ...
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