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Primer, kit and method for detecting PIGA gene promoter region 4972A>G mutation site

A PIGA-4972-F and PIGA-4972-R technology, which is applied in the field of primers for detecting the 4972A>G mutation site in the promoter region of the PIGA gene, can solve the problem of not being able to produce PNH phenotypes, and achieve high and low costs and the effect of difficulty and detection difficulty

Inactive Publication Date: 2019-04-16
南昌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

[0005] In addition, PIG-A protein only acts as a catalyst to participate in the synthesis of GPI, so PIG-A protein can produce a large amount of GPI with only partial activity. Therefore, many missense mutations of PIG-A gene cannot produce PNH phenotype

Method used

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  • Primer, kit and method for detecting PIGA gene promoter region 4972A>G mutation site
  • Primer, kit and method for detecting PIGA gene promoter region 4972A>G mutation site
  • Primer, kit and method for detecting PIGA gene promoter region 4972A>G mutation site

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The primers for detecting the mutation of the 4972A>G site in the promoter region of the PIGA gene are designed for the amplification primers designed for the site 4972A>G in the promoter region of the PIGA gene, including:

[0037] Primers for amplifying the 4972A>G site in the promoter region of the PIGA gene, the base sequence of which is:

[0038] PIGA-4972-F: TGTAAAACGACGGCCAGTCTCAGCGCCTCTTTGTGAT

[0039] PIGA-4972-R: AACAGCTATGACCATG ACCCTCCGACCCAACTTC.

[0040] A kit for detecting the 4972A>G site mutation in the promoter region of the PIGA gene, including

[0041] (i) Blood DNA extraction reagents;

[0042] (ii) detection system PCR reaction solution;

[0043] (iii) Sequencing system reagents;

[0044] Among them, the tissue DNA extraction reagent can be purchased from commercial reagents such as Tiangen DNA extraction kit.

[0045] Detection system PCR amplification reaction solution includes: 2×PCR Buffer; 2mM dNTPs; KOD FX DNA Polymerase (1U / μl); upstrea...

Embodiment 2

[0048] Operation process of blood / cell / tissue genomic DNA extraction kit (Tiangen Biology):

[0049](1) Extract tissue DNA from blood: 1) Extract 300 μl of blood and add 900 μl of erythrocyte lysate, mix by inverting, and place 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 i...

Embodiment 3

[0075] The clinical samples of 2 cases were extracted genome, prepared reagents, amplified and sequenced according to the reagents and methods of Examples 1 and 2. Add 1 μl of sample to each detection system PCR reaction solution. Electrophoresis results such as figure 1 As shown, it shows that the primer PIGA-4972-F / R of the present invention can effectively amplify the blood sample, and the band is single.

[0076] The test results of samples 1 and 2 are shown in 2:

[0077] figure 2 It shows the sequence screenshots of the 4972A>G site mutation in the PIGA gene promoter region of samples 1 and 2, indicating that the 4972A>G mutation occurred in sample 1.

[0078] It can be seen from the detection results that the primers of the present invention can amplify the 4972A>G site sequence in the promoter region of the PIGA gene, and the sequencing results are completely accurate, regardless of whether it is a wild type or a mutant type.

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Abstract

The invention discloses a primer for detecting a PIGA gene promoter region 4972A>G mutation site. The base sequences of the primer are PIGA-4972-F:TGTAAAACGACGGCCAGTCTCAGCGCCTCTTTGTGAT and PIGA-4972-R:AACAGCTATGACCATGACCCTCCGACCCAACTTC. By means of a Sanger sequencing technology, the mutation site can be quickly discovered, the detection result is accurate, the primer can assist in detecting the mutation situation of a region, and a reference significance is provided for investigation of causes of diseases.

Description

technical field [0001] The invention belongs to the field of molecular detection, in particular to a primer, a kit and a detection method for detecting the 4972A>G mutation site in the PIGA gene promoter region. Background technique [0002] The PIG-A gene is located on chromosome Xp22.1. The full length is about 17kb, encoding a protein product containing 484 amino acids. The coding region consists of 1452bp. A total of 6 exons (such as figure 1 shown). Exon 1 contains part of the 5' untranslated region. Exon 2 contains the rest of the 5'untranslated region and about half of the coding region, the 3'untranslated region is located at the 3' end of exon 6 (about 2.1kb), between exon 1 and its upstream 583bP Between them is the promoter region, in which there are 4 CAAT boxes, 2 AP-2 sequences, and 1 cis-regulatory element. These sequences are very important for the expression of the PIG-A gene. [0003] The PIG-A gene is involved in the first step of GPI biosynthesi...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858
Inventor 刘赵玲吴鹏飞王淑一
Owner 南昌艾迪康医学检验实验室有限公司
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