Method for detecting multiple endocrine adenoma II gene mutation

A gene and mutation technology, applied in biological testing, material inspection products, etc., can solve the problems of low incidence of MEN2, different RET gene mutation sites, and no RET gene mutation status

Inactive Publication Date: 2008-03-26
RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

And for different regions, different human groups, and different human races, the mutation sites of the RET gene are not the same, and the differences are large
Especially in China, due to the low incidence of MEN2 and the need for a lot of work and time to collect and summarize, there are no research reports on the status of RET gene mutations in the Chinese population
[0008] Moreover, in the current prior art, the process of detecting the mutation of the MEN2 pathogenesis-associated site of the RET gene is very complicated and the site is not clear, and the inspection time usually lasts for several days, and this aspect also needs to be improved

Method used

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  • Method for detecting multiple endocrine adenoma II gene mutation
  • Method for detecting multiple endocrine adenoma II gene mutation
  • Method for detecting multiple endocrine adenoma II gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] The RET gene mutation research of embodiment 1MEN2A

[0113] 1. Object

[0114] A total of 15 MEN2A proband cases were collected from January 2002 to February 2006. These cases came from different regions, including: Shanghai, Zhejiang, Jiangsu, Fujian, Guangdong, Jiangxi, Shandong, Shaanxi, Sichuan, Liaoning, etc. . The family members of each proband were investigated for clinical history, blood biochemistry and DNA specimens were collected. A total of 119 samples were collected.

[0115] 2 RET proto-oncogene detection in the proband and family members

[0116] 2.1 Extraction of peripheral blood genomic DNA (phenol-chloroform extraction method).

[0117] 5ml of whole blood from the median forearm vein of the proband and family members of 119 people was collected, placed in a 15ml centrifuge tube containing 1ml of ACD anticoagulant, and turned upside down several times to fully mix the blood and anticoagulant. Specific steps:

[0118] (1) Add 8 ml of ice-precooled...

Embodiment 2

[0166] The RET gene mutation research of embodiment 2MEN2B

[0167] 1. Object

[0168] From April 2002 to September 2005, 5 MEN2B families were collected from all over the country, including 5 probands and 23 family members.

[0169] 2RET proto-oncogene detection

[0170] 2.1 Extraction of genomic DNA from peripheral blood

[0171] Collect 5ml of peripheral anticoagulant blood from the proband and family members, and use the U-gene Blood DNA Kit to extract the blood genomic DNA. The specific steps are:

[0172] (1) Add 250ul whole blood sample into a 1.5ml centrifuge tube.

[0173] (2) Add 250ul of XY buffer solution (U-gene), shake and mix well.

[0174] (3) Add 20 ul of proteinase K (10 mg / ml), shake vigorously and mix well.

[0175] (4) Incubate at 56°C overnight, and the color will turn dark green.

[0176] (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...

Embodiment 3

[0193] Embodiment 3 detects the method for RET gene mutation

[0194] Since it is very cumbersome to carry out the RET gene mutation by taking the steps described in the foregoing Example 1 or Example 2, especially sequencing is required. The disadvantage of sequencing is that it takes a long time (about 3 days) and the process is complicated.

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Abstract

The present invention belongs to the field of biotechnology, and discloses one method of in vitro determining whether to have RET gene variation in the nucleic acid sample, one kit for determining RET gene variation and one process of obtaining RET gene amplifying product. The method of the present invention is accurate, simple, fast and stable.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention relates to a method for obtaining RET gene amplification products, and the amplification products can be used to detect RET gene variation without sequencing. Background technique [0002] The incidence of multiple endocrine neoplasia type 2 (MEN2) is about 1 / 30,000, and it is divided into MEN2A, MEN2B and familial medullary thyroid carcinoma (FMTC). Almost all patients with MEN2A have medullary thyroid carcinoma (MTC, a malignancy arising from calcitonin-secreting C cells) or C-cell hyperplasia. Pheochromocytoma and parathyroid hyperplasia account for approximately 50% and 15-30% of MEN2A patients, respectively. In addition to MTC, 50% of MEN2B patients showed pheochromocytoma, and a smaller part showed intestinal ganglioma, corneal nerve enlargement and Marfan signs. MTC is the only phenotype in FMTC patients. [0003] Many MEN2 diseases are caused by RET gen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N33/68
Inventor 宁光崔斌赵咏桔王卫庆周瑜琳
Owner RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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