ABSTRACT
Objective To construct human yippee-like 5(YPEL5) gene eukaryotic expression recombinant plasmid and to express in esophageal carcinoma EC9706 cells .Methods The cDNA from human normal tissue was taken as a template and amplified to YPEL5 gene coding sequence with 366 bp in length .Then this se-quence was inserted into the multiple cloning site areas of eukaryotic expression vector pCDH-CD513B for ob-taining the eukaryotic expression vector pCDH-CD513B-Flag-YPEL5 .After the bacterial colony PCR identifi-cation ,it was sent to the corporation for testing the sequence .The successfully constructed recombinant plas-mid was transfected into human esophageal carcinoma EC9706 cells .The expression of PEL5 gene in EC9706 cells was detected by QRT-PCR and Western Blot .Results The YPEL5 gene segment with 366 bp in length was successfully amplified .pCDH-CD513B-Flag-YPEL5 recombinant plasmid was obtained by double enzyme digestion ,connection ,conversion and screening .The gene sequencing identification showed that the inserted gene sequence in recombinant plasmid was consistent with that in the GenBank .After 2 d of transfecting into EC9706 cells ,the QRT-PCR and Western Blot revealed that YPEL5 gene expression was significantly up-reg-ulated .Conclusion The pCDH-CD513B-Flag-YPEL5 eukaryotic expression vector is successfully constructed and is expressed in esophageal squamous cancer cell line EC9706 ,thus which lays a foundation for studying its function in the progression of esophageal cancer .
ABSTRACT
ObjectivesTo investigate the clinical value of the melting curve analysis-based PCR assay for the clinical genetic diagnosis and prenatal diagnosis of β-thalassemia.Methods A total of 451 peripheral blood samples,including 372 cases with β-thalassemia phenotypes and 79 cases without β-thalassemia phenotypes,were collected by Liuzhou Municipal Maternity and Child Healthcare Hospital between January 2011 and August 2011.Moreover,another 84 cases,including 16 fetal villi samples (10 - 13 weeks),64 amniotic fluid samples (16 -24 weeks ) and 4 umbilical cord blood samples (above 17 weeks),whose parents were β-thalassemia carriers,were also collected for this assay between June 2011 and September 2011.A double-blind test was done to compare the detection reliability of the melting curve analysis-based assay (24 β-thalassemia mutations can be detected) with PCR-RDB probe assay (17 β-thalassemia mutations can be detected ) and DNA sequencing using these samples.The wildtype,mutant and total concordance rates of the genotyping results were calculated separately among the melting curve analysis based assay,PCR-RDB probe assay and DNA sequencing.Results Among the 451 peripheral blood samples,thirteen mutations and nineteen genotypes were obtained by using melting curve analysis-based assay.447 samples had the same detection results and 4 samples had different detection results by comparing melting curve analysis-based assay with PCR-RDB probe assay,thus,the concordance rate of the sample detection result was 99.1％ (447/451),and the concordance rate of the allele detection result was 99.6％ (898/902).DNA sequencing results of the 4 samples showed that 3 samples had the same genotyping result with melting curve analysis-based assay,and 1 sample had the same genotyping result with PCR-RDB probe assay.A rare β-globin mutation which was not included by melting curve analysis-based assay was not detected.Thus,the genotypes of 450 samples were detected accurately by melting curve analysis-based assay,and the concordance rate of the sample detection between the melting curve assay and DNA sequencing assay was 99.8％ (450/451).Among 84 fetal villi,amniotic fluid,and umbilical cord blood samples,8 mutation types and 18 genotypes were obtained by using melting curve analysis-based assay.All the samples have the same detection results by comparing melting curve analysis-based assay with PCR-RDB probe assay and DNA sequencing,so the concordance rate of the genotyping results was 100％ among the melting curve analysis-based assay,PCR-RDB probe assay and DNA sequencing.Conclusions The melting curve analysis-based PCR assay can detect multiple unknown samples simultaneously,and detect multiple mutations accurately.It is very useful for the genetic diagnosis and prenatal diagnosis of β-thalassemia.