A method and primers for detecting all exons of an IL7R gene

An all-exon, sequencing primer technology, used in life sciences and biology, can solve problems such as weakening the effect of steroid hormone therapy

Inactive Publication Date: 2018-04-10
北京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

Current research has found that IL7R has mutations at multiple sites in pediatric T-ALL, such as S185C, V253G, and exon insertions and deletions, etc. These mutations in IL7R can affect the treatment of pediatric T-ALL through the IL7R/

Method used

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  • A method and primers for detecting all exons of an IL7R gene
  • A method and primers for detecting all exons of an IL7R gene
  • A method and primers for detecting all exons of an IL7R gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] A primer for detecting the polymorphic mutation site of IL7R gene, the design of the primer is the amplification primer designed for the whole exon of IL7R, including:

[0092] The primers for amplifying the whole exon sequence of IL7R gene, its base sequence is:

[0093] IL7R-Exon1-F TGTAAAACGACGGCCAGTGGCTCTGTCTCTTCATGTCCTTG

[0094] IL7R-Exon1-R AACAGCTATGACCATGTGACTCAGGTCTGTTAGGGAACTG

[0095] IL7R-Exon2-F TGTAAAACGACGGCCAGTGTCTGCCACAGAGTCTGCTATT

[0096] IL7R-Exon2-R AACAGCTATGACCATGGCTGAACCGTGAGAGGAGATT

[0097] IL7R-Exon3-F TGTAAAACGACGGCCAGTTTCCTGAACATGCCTCCACTC

[0098] IL7R-Exon3-R AACAGCTATGACCATGTAACCTACAGAATGTCCAGACACA

[0099] IL7R-Exon4-F TGTAAAACGACGGCCAGTCTGGAGAATGCGGACTGGAT

[0100] IL7R-Exon4-R AACAGCTATGACCATGTGCCTGATTTGCCTATGAAAGTG

[0101] IL7R-Exon5-F TGTAAAACGACGGCCAGTCACTCTCCCTCCTTGACCACTC

[0102] IL7R-Exon5-R AACAGCTATGACCATGCTCTCACTTGCTCCCACACTT

[0103] IL7R-Exon6-F TGTAAAACGACGGCCAGTCAAAGCACCCTGAGACCCTAC

[0104]IL7R-Exon6-R AACAGCT...

Embodiment 2

[0119] Bone Marrow / Blood / Tissue Genomic DNA Extraction Kit (Tiangen Biological) operation process:

[0120] (1) Extract tissue DNA from blood: 1) Extract 300 μL of bone marrow or blood and add 900 μL of erythrocyte lysate, mix by inversion, leave at room temperature for 5 minutes, and mix by inversion several times during the period. 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 solution GB, mix thoroughly by inversion, place at 70°C for 10 minutes, the solution should become clear, and centrifuge briefly to remove water droplets on the inner wall of the tube cap. 4) Add 200 μL of absolute ethanol, shake and mix well for 15 seconds, at this time flocculent precipitation may appear, and centrifuge briefly to remove the water droplets in the tube cap. 5) Add the solution and flocculent precipitate obtained ...

Embodiment 3

[0159] Take 16 cases of clinical samples (numbered ex1-ex8 according to the primers), and extract genomic DNA, prepare reagents, amplify and sequence according to the reagents and methods of Examples 1 and 2. Add 1 μL of the sample to the detection system PCR reaction solution. Electrophoresis results such as Figure 1-a to Figure 1-h As shown, it shows that the primers of the present invention can effectively amplify blood samples, and the band is single.

[0160] The partial sequencing results of the samples are as follows: figure 2 Shown: (a) shows the wild-type sequencing screenshot of exon 5 of IL7R gene in sample No. 1, indicating that no mutation occurs in exon 5 of IL7R in the sample; Screenshot of the wild-type sequencing of the offspring, indicating that the IL7R exon 6 of the sample is not mutated;

[0161]It can be seen from the detection results that the primers of the present invention have included exon sequences, can amplify all exons of the IL7R gene, and ...

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Abstract

Primers and a method for detecting all exons of an interleukin-7 receptor (IL7R) gene related to T-cell acute lymphoblastic leukemia (T-ALL) are disclosed. The primers include eight pairs of primers for application detection of all exon sequences. A Sanger sequencing technique and sequencing primers are adopted. The disclosed primers and the method can rapidly detect all exon sequences and relatedmutants of the IL7R gene. A detection result obtained by utilizing the primers and the method is accurate, can provide a basis for T-ALL treatment, and is of great significance for selection of individual treatment schemes.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and particularly relates to a primer and a method for detecting IL7R gene mutation related to acute T lymphocytic leukemia. Background technique [0002] Acute lymphoblastic leukemia (ALL) in children is the most common neoplastic disease in children. More than 80%. Children's T-cell acute lymphoblastic leukemia (T-cell acute lymphoblastic leukemia, T-ALL) is a high-risk type of ALL, accounting for about 15%-25% of ALL, with a high recurrence rate and extremely poor prognosis, seriously affecting children life and health. Recent studies have found that the incidence of interleukin-7 receptor (IL7R) gene mutations in children with acute T-lymphoblastic leukemia is as high as 10%, which may be one of the key factors affecting the development of T-ALL in children. [0003] IL7R is a transmembrane receptor belonging to the type I cytokine receptor family, and is an essential factor in...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2531/113C12Q2535/101
Inventor 牛林梅吴鹏飞王淑一
Owner 北京艾迪康医学检验实验室有限公司
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