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Detection method of gene mutation of mitochondrial succinate dehydrogenase and kit

A technology of succinate dehydrogenase and mitochondria, applied in the field of detection methods and kits of mitochondrial succinate dehydrogenase gene mutations, can solve problems such as high complexity and difficulty in obtaining gene fragments

Inactive Publication Date: 2010-03-31
RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, PCR technology is still the most commonly used technology for the detection of gene variation. However, for the amplification of SDHB and SDHD, since the genomic DNA of the blood sample of the individual to be tested is usually used as a template, the complexity is high, and it is difficult to obtain an ideal PCR method. The target gene fragment

Method used

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  • Detection method of gene mutation of mitochondrial succinate dehydrogenase and kit
  • Detection method of gene mutation of mitochondrial succinate dehydrogenase and kit
  • Detection method of gene mutation of mitochondrial succinate dehydrogenase and kit

Examples

Experimental program
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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

[0137] The optimization of embodiment 2 PCR amplification conditions

[0138] 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.

[0139] 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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Abstract

The invention discloses a reagent for detecting whether gene mutation of mitochondrial succinate dehydrogenase (SDH) exists in samples and a kit containing the reagent. The reagent or the kit can be used for susceptibility analysis of pheochromocytoma. The invention also discloses an optimized method by which SDH gene amplification products are obtained and a method for determining whether gene mutation of SDH exists in the nucleic acid samples to be tested.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention relates to a method and a kit for detecting the mutation of the mitochondrial succinate dehydrogenase (SDH) gene. Background technique [0002] Familial paragangliomas of the head and neck are benign tumors of nonchromaffin tissues of the head and neck, most commonly located in the carotid body. As chemoreceptors, the carotid body can sense the hypoxic stimulation in the circulation, and then produce a series of systemic responses, and chronic hypoxia can lead to carotid body cell hyperplasia. The adaptive response of mammalian cells to hypoxia depends on the function of mitochondria, by increasing the production of reactive oxygen species (ROS) and then activating erythropoietin ( EPO), vascular endothelial growth factor (VEGF), and transcription of glycolytic enzymes, ultimately increasing oxygen delivery and promoting glycolytic ATP production. Thus, mitochon...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12N15/10
Inventor 宁光崔斌王卫庆郑旭磊苏颋为姜蕾周薇薇袁文祺
Owner RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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