Reagent and method for detecting leucocythemia susceptibility

A kit and susceptibility technology, applied in biochemical equipment and methods, microbial assay/inspection, etc., can solve problems such as expression decline

Inactive Publication Date: 2006-01-25
PEKING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, the National Human Genome South Research Center used DNA chip technology to study differentially expressed genes in liver cancer and found that the expression of some apoptosis-related genes such as PDCD5, PDCD8, Bak, TRAF6 and TRAIL was significantly decreased in liver cancer

Method used

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  • Reagent and method for detecting leucocythemia susceptibility
  • Reagent and method for detecting leucocythemia susceptibility
  • Reagent and method for detecting leucocythemia susceptibility

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1. Obtaining SNP

[0030] Step 1: Extraction of genomic DNA

[0031] Using a small amount of peripheral blood leukocyte genomic DNA rapid extraction and purification kit (Shanghai Huashun Biological Engineering Co., Ltd.), 2ml of human whole blood was used to extract genomic DNA; after the concentration was adjusted to 50ng / μl, it was used for conventional PCR amplification.

[0032] Step 2: Obtain SNP

[0033] The genomic DNA extracted in step 1 is subjected to specific PCR amplification, and the amplified product is purified and then directly sequenced. The sequence of each PCR amplification product determined is compared to obtain the sequence difference and obtain the SNP. Among them, the PCR reaction conditions were 95°C for 3 minutes, 35 cycles of 95°C for 30 seconds, 68°C for 1 minute, and finally 72°C for 7 minutes. The primers are:

[0034] Sense primer: 5'-CGGGGAATCGGGCCTCTGC-3' (SEQ ID NO: 2)

[0035] Antisense primer: 5'-CAAGCTCCTCGTCCGCCATG-3' (SEQ ID N...

Embodiment 2

[0037] Example 2. Detection kit

[0038] Step 1: Extraction of DNA template

[0039] 2ml of human peripheral blood was drawn by conventional methods, and genomic DNA in these blood samples was extracted by conventional methods.

[0040] Step 2: PCR reaction

[0041] Prepare a detection kit for detecting the susceptibility of PDCD5 gene-related diseases, which contains the following primer pairs that can amplify 154 and 170 SNPs:

[0042] Sense primer: 5'-GCTCCGGGCTGGATTGGTG-3' (SEQ ID NO: 6)

[0043] Antisense primer: 5'-CATGGCTCGGCGTCAGCG-3' (SEQ ID NO: 12)

[0044] The PCR reaction conditions were 95°C, 3 minutes, 35 cycles of 95°C for 30 seconds, 68°C for 1 minute, and finally 72°C for 7 minutes.

[0045] Step 3: Genetic analysis

[0046] The amplified product was genotyped with RFLP technology. Because the 170th G polymorphism produced a Nar I endonuclease site and the 154th A destroyed a Nar I restriction site, the Nar I enzyme was used for the analysis. Enzyme digestion can ...

Embodiment 3

[0047] Example 3. Analysis of the functional effects of the 154A>G / 170G>A polymorphism in the PDCD5 promoter region Step 1. Construction of the reporter vector for the 154G / 170A genotype and the promoter region of the 154A / 170G genotype

[0048] A 743 bp DNA fragment containing the 154G / 170A mutant genotype and the 154A / 170G wild genotype of the PDCD5 promoter region from the 643th position upstream of the transcription start site to the 91 position downstream of the transcription start site was cloned into pGL3-Basic( Promega company) report plasmid, construct the luciferase reporter gene plasmid pGL3-PDCD5-170A (154G / 170A) containing 154G / 170A mutant type and pGL3-PDCD5-170G (154A / 170G) containing 154A / 170G wild type pGL3-PDCD5-170G (154A / 170G) Luciferase reporter gene plasmid.

[0049] Step 2. Detection of promoter activity of 154G / 170A genotype and 154A / 170G genotype

[0050] The negative control pGL3-Basic, the positive control pGL3-SV40 (Promega), pGL3-PDCD5-170A and pGL3-PD...

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Abstract

The invention discloses a agent box to testing leucocyte susceptibility that includes two SNPs specificity primer in the 154th and the 170th of the upstream 5' adjust region SEQ ID NO: 1 that is testing PDCD5 gene, and restrain inscribe enzyme that is testing the SNPs site. The invention predicts the susceptibility of leucocyte by testing the site of SNP.

Description

Technical field [0001] The present invention relates to a kit for disease detection, in particular a kit for detecting leukemia susceptibility, by detecting two completely linkage disequilibrium functional single nucleotide polymorphisms in the promoter region of human PDCD5 gene ( SNP) sites are used to predict the susceptibility of individuals to leukemia, and further involve methods for detecting susceptibility to leukemia. Background technique [0002] Programmed cell death (programmed cell death) is a physiological cell self-destruction process, which plays a very important role in embryonic development, the stability of the biological environment, and the defense of multicellular organisms against external and internal damage. The disorder of the apoptosis process may be directly or indirectly related to the occurrence of many diseases, such as tumors, autoimmune diseases, neurodegeneration such as Alzheimer's disease and local injuries. The research of apoptosis-related mo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 赵红珊马曦阮国瑞马大龙王莹李启艳朱平
Owner PEKING UNIV
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