Detection method of gene mutation of mitochondrial succinate dehydrogenase and kit
A succinate dehydrogenase and mitochondrial technology is applied in the field of mitochondrial succinate dehydrogenase gene mutation detection methods and kits, which can solve the problems of high complexity and difficulty in obtaining gene fragments.
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Embodiment 1
[0088] Detection of gene mutations in patients with sporadic pheochromocytoma using optimized primers
[0089] 1. Obtain the genomic DNA of the blood sample of the test subject
[0090] Collect 5ml of peripheral anticoagulated blood from patients with sporadic pheochromocytoma, and use U-gene Blood DNAKit to extract blood genomic DNA. The specific steps are:
[0091] (1) Add 250 μl whole blood sample into a 1.5ml centrifuge tube.
[0092] (2) Add 250 μl of XY buffer solution (U-gene), shake and mix well.
[0093] (3) Add 20 μl of proteinase K (10 mg / ml), shake vigorously and mix well.
[0094] (4) Incubate overnight at 56°C.
[0095] (5) Add 260 μl of absolute ethanol, shake to mix, centrifuge at 12,000 rpm for 1 minute, and shake the solid precipitate in the solution to the bottom of the tube.
[0096] (6) Put the DNA separation column in a 2ml collection tube, pour the solution obtained in the previous step into the column, centrifuge at 8000rpm for 1min, and discard the c...
Embodiment 2
[0129] The optimization of embodiment 2 PCR amplification conditions
[0130] Using the blood sample genomic DNA of a patient with sporadic pheochromocytoma as a template, the primers shown in Table 1 were used to amplify SDHB and SDHD exons respectively. The PCR amplification method and conditions were the same as in Example 1, and the obtained PCR The product was subjected to agarose gel electrophoresis, and the electrophoresis results were shown in Figure 1-2 . It can be seen that each PCR reaction product is a band of a single target gene fragment, and the band brightness is strong, obvious and clear, and there is no miscellaneous band.
[0131] Also using the blood sample genomic DNA of the aforementioned patient with sporadic pheochromocytoma as a template, the primers listed in Table 2 below (the primers are designed according to the sequences of SDHB and SDHD but different from the primers in Table 1) were used for PCR amplification. The amplification method and con...
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