| Objective:1.To establish a SNP typing method based on melting curve analysis of DNA fragments of different lengths,and then type rs4946936,rs2253310,rs2802288,rs12212067 and rs2802292 in the non-coding region of human FOXO3A related to life span,oxidative stress and metabolism,etc.2.To construct plasmid standards,and then establish FOXO3A gene expression quantitative detection system.3.To genotype the The SNPs of different individuals and quantitative FOXO3A gene expression levels,and then analyze the effect of each SNP type on FOXO3A gene expression.Methods:1.Selected 14 healthy blood donors and isolated the peripheral blood mononuclear cells(PBMCs)using Human peripheral blood lymphocyte separation fluid,Genomic DNA(gDNA)and total RNA(total RNA)were Extracted.2.Established McSNP typing method.Firstly,designed a specific amplification primer containing SNP site,amplified the target gene by PCR,digested the PCR product with restriction enzyme.Finally,the digestion products were analyzed by melting curve,typed the SNP.Verified the accuracy of the typing method by PCR-restriction analysis(PCR-RFLP)and PCR product sequencing.3.Established the FOXO3A gene expression quantitative system.Firstly,constructed the recombinant plasmids pGM-T-FOXO3A and pGM-T-GAPDH by T-A cloning method,obtained plasmid standards by diluted the recombinant plasmids.Established the standard curve by amplificating these standards.Designed specifically for the quantitative amplification of FOXO3A gene amplification primers,different concentrations of RNA samples repeated repeated quantitative detection,intra-batch and inter-batch precision and accuracy were calculated to test the reliability of quantitative detection system.4.The SNP typing and FOXO3A gene expression were quantified in 10 DNA and RNA samples from different individuals.SPSS statistical software was used to compare the differences in gene expression between groups,and an independent sample T-test was performed.Results:1.The McSNP genotyping results of the 14 blood donors rs4946936 and 5 blood donors rs2802288 were all consistent with those obtained by the RFLP genotyping method.The sequencing of the PCR products also confirmed the accuracy of the above genotyping results.the results of McSNP typing method of rs4946936 of TT type showed a single peak with a Tm value of 77.25°C,TC type showed double peaks with a Tm value of 77.25°C and 71.75°C,and CC type showed a Tm value of 71.25°C.peak.2.TA clone blue-white screening experiments showed that the plasmid was ligated and transformed successfully,and the positive clones were identified by PCR,which showed that the plasmid contained the target fragment.he extracted standard plasmid as a standard dilution of the standard curve produced by the amplification efficiency of0.9-1.1,the correlation coefficient greater than 98%.Quantitative analysis of FOXO3A gene using different plasmids as standard samples showed that the coefficient of variation(CV)was less than 5%and the bias was less than 7.5 in the range of 1.56×10~6-1.56×10~3cops/?l(Ct value of 20-30)%,Test results have high credibility.3.Among the 10 samples used for the analysis of differences in gene expression among different SNP genotypes,rs4946936 has 1 TT genotype,3 TC genotypes and 6CC genotypes.Compared with CC group,rs4946936T mutant group(TT and TC type)showed no significant difference in FOXO3A gene expression(p>0.05).Conclusion:1.Successfully established a FOXO3A SNP typing method based on lysing curve analysis.2.The establishment of FOXO3A gene quantitative detection system,the system of FOXO3A gene expression with high precision and accuracy.3.Analysis of 10 samples from healthy people did not find significant differences in FOXO3A gene expression between individuals with different rs4946936 genotypes. |
PDF Full Text Request