| Objective:To explore the application of mutation sensitivive molecular switches technology platform detecting G389A and 968delA of PTEN gene mutations in the screening of tissue DNA in patients with endometrial cancer,and analysis the correlation between the PTEN mutations and occurrence and development of endometrial cancer.Methods:Firstly,96 normal blood DNA samples was extracted as templates,and we design two primers that have complementary overlapping area,which can be amplified exon 5 fragment and exon 8fragment of PTEN gene respectively.Secondly,the two fragments by overlapping PCR method were connected to get the wild PTEN gene fusion segments,then insert these fragments into pMD19-T carrier to get a wild type pMD19-exon 5-exon 8 fusion restructuring carrier.The wild type recombinant plasmid as a template was used for designing the primers including G389A and 968delA.According to the principle of overlapping PCR site-directed mutagenesis,we get PTEN gene fusion segments containing G389A and 968delA mutations,and connect with the T vector to get mutant recombinant plasmid,too.The two plasmids were screened positive clones by microbial PCR and a blue and white screen,then were verified for DNA sequencing.Wild-type specific primers and mutant-type specific primers by the templates constructed recombinant plasmids were designed respectively with 3'terminal phosphorothioate modification.Under the high fidelity of DNA polymerase,molecular switch detect the two templates and mutation-type templates with G389A or 968delA mutations,and explore this technology of the sensitivity and specificity of detection.Thirdly,the technology platform that established molecular switch were used for screening G389A or 968delA mutations of PTEN gene in tissue DNA with endometrial carcinoma.Finally,we analysis the correlation between two loci mutations of PTEN gene and endometrial cancers in China.Results:Microbial PCR and DNA sequencing confirmed wild-type and mutant-type plasmid templates harboring 389 and 968 two sites of PTEN gene were successfully established by overlapping PCR in vitro.G389A and 968delA two loci of PTEN gene were tested successfully by molecular switch technology,and the electrophoresis results showed that when the allele specific detection primer?wild type/mutant type?matches plasmid templates?wild type/mutant type?completely,there will be a corresponding strip with PCR products,Conversely,the mutations sensitive molecular switch can't extend the specific primers when they matches incompletely,and there are not PCR products.What's more,the molecular switch can be used for gene mutation detection when the mutant template's concentration is greater than 10-3 ng/?L.In the screening of G389A and 968delA two mutations of PTEN gene in the patients with 21 normal endometrium and 62 patients with endometrial carcinoma of tissue DNA respectively,we found that 1 case of 389 G>A of PTEN gene mutations were detected in EC samples,but968delA was not detected,and this results were confirmed by sequencing.The GG genotype frequency of EC group was 0.984,and the mutated GA or AA genotype frequency was 0.016,higher than that of normal control GA or AA genotype frequency?P>0.05?.Conclusions:The mutations sensitive molecular switch technology platform were successfully established to detect G389A and 968delA two loci mutations of PTEN gene.And this technology was successfully applied to screening the mutations of PTEN gene in tissue DNA with endometrial cancer that had been not reported in the domestic.Finally through the analysis of the genotype frequencies of 389 G>A we found that the morbidity of endometrial carcinoma in China have no relationship of G389A and 968delA of PTEN gene,which the results can provide a new thought for clinical diagnosis and treatment of endometrial carcinoma. |
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