Reagent and method for detecting leucocythemia susceptibility
A kit and susceptibility technology, applied to the kit for detecting leukemia susceptibility, in the field of detecting leukemia susceptibility, can solve the problem of expression decline
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Embodiment 1
[0029] The acquisition of embodiment 1.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 Bioengineering Co., Ltd.), 2ml of human whole blood was used to extract genomic DNA; after the concentration was corrected to 50ng / μl, it was used for conventional PCR amplification.
[0032] Step 2: SNP acquisition
[0033] Perform specific PCR amplification on the genomic DNA extracted in step 1, perform direct sequencing after purification of the amplified products, and compare the sequences of the PCR amplification products determined to obtain sequence differences and obtain SNPs. Wherein, the PCR reaction condition is 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. Primers are:
[0034] Sense primer: 5'-CGGGGAATCGGGCCTCTGC-3' (SEQ ID NO: 2)
[0035] Antisense primer: 5'-CAAGCTCCTCGTCCGCCATG-3'...
Embodiment 2
[0037] Embodiment 2. Detection kit
[0038] Step 1: Extraction of DNA template
[0039] 2 ml of human peripheral blood was extracted 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 the 154-position and 170-position SNP:
[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. for 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: Gene Analysis
[0046] Genotype analysis was carried out on the amplified product by RFLP technology. Since the 170th G polymorphism produced a Nar I endonuclease site and the 154th A destroyed a Nar I restriction site, ...
Embodiment 3
[0047] Example 3. Functional effect analysis of 154A>G / 170G>A polymorphism in the PDCD5 promoter region Step 1, Construction of 154G / 170A genotype and 154A / 170G genotype promoter region reporter vector
[0048] A 743bp DNA fragment from position 643 upstream of the transcription start site to position 91 downstream of the transcription start site in the PDCD5 promoter region containing the 154G / 170A mutant genotype and the 154A / 170G wild genotype was cloned into pGL3-Basic ( Promega Company) reporter plasmid, the luciferase reporter gene plasmid pGL3-PDCD5-170A (154G / 170A) containing 154G / 170A mutant type and the pGL3-PDCD5-170G (154A / 170G) containing 154A / 170G wild type were constructed. Luciferase reporter gene plasmid.
[0049] Step 2. Detection of 154G / 170A genotype and 154A / 170G genotype promoter activity Transfect negative control pGL3-Basic, positive control pGL3-SV40 (Promega), pGL3-PDCD5-170A and pGL3-PDCD5-170G respectively In HEK293 cells, the cells were collected ...
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