Detection Of β-thalassemia Mutations By Proofreading Polymerase-mediated Mutation-sensitive On/off Switch

Objective：To detect β-thalassemia mutations proofreading polymerase-mediatedmutation-sensitive on/off switchMethods： Genomic DNA was extracted from blood samples of normalindividuals.According to the known hot spot mutations in β-globin gene,designedmutant-specific primers were designed.To prepare mutation templates,primerextension reaction was performed with low-fidelity enzyme,and products harboringmutations were cloned into T vector as template for further experiments.We haveconstructed six types of clones of β-thalassemia mutations:CD41-42、IVS-2nt654、TATAbox-28、CD17、CD71-72、CD26.In assay development,both the cloned plasmidand genomic DNA were used as the template respectively.Two directional primerextension was performed using mutation-sensitive on/off switch in differentsystems.Thereafter,multiplex primer-extension was developed.Finally,patient DNAsamples respectively containing CD26and TATAbox-28mutations were analyzed.Result： Six hot spot mutations were respectively detected in differentsystems.When normal chromosomal DNA samples were used,whereas the wild-typeallele primers can be extended,the mutant alleles of the locus-specific primer can notbe extended. Conversely,with the use of the mutant plasmid template,the mutantalleles primers were extended,but the wild type alleles specific primers could not beextended.Simultaneous detection of six hot spot mutations in the same reactionsystem was established.When the primer is fully match with the template,the primer isextended;when the primer is not fully match with the template,the primer is notextended.Conclusion： An assay with high-fidelity polymerase-mediated mutation-sensitiveon/off switch for rapid screening of β-thalassemia gene hotspot mutation has beenestablished and may have clinical applications in genetic diasgnosis and epidemical screening of the related diseases.