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Primers, kit and method for detecting mutation of intron 4 in gene ABCG8

A technology of introns and sequencing primers, applied in the fields of life sciences and biology, can solve problems such as mutations that have not been reported, and achieve the effects of high cost, low cost and difficulty, and difficult detection

Inactive Publication Date: 2018-12-07
合肥艾迪康医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Since there are no hotspot mutations in ABCG8, and there are many mutations that have not been reported

Method used

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  • Primers, kit and method for detecting mutation of intron 4 in gene ABCG8
  • Primers, kit and method for detecting mutation of intron 4 in gene ABCG8
  • Primers, kit and method for detecting mutation of intron 4 in gene ABCG8

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Extraction of DNA in blood: 1) Extract 300 μl of blood and add 900 μl of erythrocyte lysate, mix by inversion, and let stand at room temperature for 5 minutes, during which time, invert and mix several times. Centrifuge at 12,000rpm for 1min, suck off the supernatant, leave the white blood cell pellet, add 200μl buffer GA, shake until thoroughly mixed. 2) Add 20 μl proteinase K solution and mix well. 3) Add 200 μl of buffer GB, mix thoroughly by inversion, place at 70°C for 10 minutes, the solution should become clear, and briefly centrifuge to remove water droplets on the inner wall of the tube cap. 4) Add 200 μl of absolute ethanol, vortex and mix well for 15 seconds. At this time, flocculent precipitates may appear. Briefly centrifuge to remove water droplets on the inner wall of the tube cap. 5) Add the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3 (the adsorption column is placed in a collection tube), centrifuge a...

Embodiment 2

[0035] PCR amplification:

[0036] The PCR amplification system reagent preparation method is as follows:

[0037]

[0038] The reaction conditions are as follows:

[0039]

[0040] Among them, the primer sequence is:

[0041] ABCG8-intron4-F:TGTAAAACGACGGCCAGTCACCTCTCTAGGGGCTGGTC

[0042] ABCG8-intron4-R: AACAGCTATGACCATGCGGAAGGCAAGCTGAGTTGT.

Embodiment 3

[0044] Purification of PCR products: Enzyme purification was configured as follows according to the system: CIP (NEB Company) 0.1 μl, ExoI (NEB Company) 0.5 μl, deionized water 1.4 μl; finally add 9 μl of PCR product. The purification reaction procedure is as follows: 50 min at 37°C, 5 min at 95°C.

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Abstract

The invention discloses primers, kit and method for detecting the 12902T>C mutation in an intron 4 region of a phytosterolemia-related gene ABCG8. Through a Sanger sequencing technology, a mutation site in this region can be quickly found. The discovery of the mutation site facilitates the determination and analysis whether the ABCG8 gene mutation causes phytosterolemia and helps to identify the cause of the disease.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and particularly relates to a primer for detecting the mutation of intron 4 of plant sterolemia ABCG8 gene, which can be used to quickly detect the mutation of intron 4 of ABCG8 by adopting common PCR technology. Background technique [0002] The ABCG8 gene is a member of the ATP-binding cassette (ABC) transporter superfamily. The protein encoded by the gene ABC protein works by transporting various molecules across cell membranes and intracellular membranes. The ABC genes are divided into seven distinct subfamilies (ABC1, MDR / TAP, MRP, ALD, OABP, GCN20, and White), and this protein is a member of the White subfamily. The function of the protein encoded by this gene is to exclude non-cholesterol sterols into the gut level, facilitate the excretion of cholesterol and sterols into bile, and enable the transport of sterols back into the intestinal lumen. It is expressed in the hepatic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/156
Inventor 桑志高吴鹏飞王淑一
Owner 合肥艾迪康医学检验实验室有限公司
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