Primer and kit for detecting mutation of CPT1A gene
A kit and gene technology, applied in the biological field, can solve the problem that there is no simultaneous determination of multiple mutations in the CPT1A gene, etc.
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Embodiment 1
[0069] Example 1: Design of Specific Amplification Primers
[0070] Design specific amplification primers according to the reference sequence of the CPT1A normal gene in the NCBI database, and the specific amplification primers should meet the following conditions:
[0071] (1) The product cannot form a secondary structure, the bases should be randomly distributed, and the primer itself cannot have 4 consecutive bases complementary;
[0072] (2) The annealing temperature difference between forward and reverse primers of all PCR fragments shall not exceed 5°C, and the annealing temperature of each product shall be between 50°C and 60°C to ensure the specificity and stability of amplification;
[0073] (3) The forward amplification primer of the PCR amplification primer is the primer used for sequencing amplification, and the length is between 18-22bp.
[0074] The specific amplification primers designed in the present invention are shown in Table 1.
Embodiment 2
[0075] Embodiment 2: sample DNA extraction
[0076] The positive sample used in the present invention was taken from a subject in Shandong Province, who was diagnosed as carnitine palmitoyltransferase 1A deficiency by the hospital, and the blood sample was taken with the consent of the subject.
[0077] Using the blood genome extraction kit (Tiangen Biochemical Technology [Beijing] Co., Ltd.) to extract genomic DNA, the exemplary steps are as follows:
[0078] (1) Add 750 μl of cell lysate CL to 300 μl of anticoagulant blood, invert and mix 5 times;
[0079] (2) Centrifuge at 12,000rpm (~13,400×g) for 1min. Discard the supernatant, and place the centrifuge tube upside down on clean absorbent paper for 2 minutes to ensure that the precipitate is in the tube (this step should be handled carefully, in order to avoid the precipitate being poured out, it is recommended to use a conical centrifuge tube). Note: In rare cases, the sediment may be very loose, so the supernatant shoul...
Embodiment 3
[0092] Embodiment 3: PCR amplification
[0093] 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.
[0094] PCR amplification conditions are: pre-denaturation at 94°C for 3min20s, denaturation at 94°C for 35s, annealing for 35s, annealing temperature for specific amplification primers at 51°C-59°C, extension at 72°C for 50s, a total of 30 cycles; final extension at 72°C for 5min .
[0095] 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.
[0096] Table 1. Specific amplification primers and annealing t...
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