The Study Of Rapid Discrimination Of Three Related Gene Mutations In AAID By On/off Switch

Objective: To rapidly detect the three hotspot mutations associated withaminoglycoside antibiotics induced deafness (AAID) by mutation sensitive molecularswitch mediated by high fidelity DNA polymerase.Methods:961delT, C1494T, A1555G hotspot mutations in mitochondrial DNA(mtDNA)12S rRNA were selected as detection target. First, normal blood genomicDNA was extracted. The amplification primers were designed according to mtDNA12S rRNA gene sequence (NC01290), and primer extension reaction was performedusing polymerases without3’exonuclease activity, then the PCR product was cloningto PMD19-T, for preparing wild type mtDNA template contains three hotspot sites.Three pairs of complementary overlapping mutagenesis primers were designed tocreate point mutations in the corresponding sites by Muta Primer2.0software basedon the sequence of the early wild-type plasmid. The wild-type plasmid was used as aPCR template, and DNA fragment including three point mutations was obtainedthrough overlap extension PCR binding three pairs of complementary overlappingmutagenesis primers and frozen precipitation, then digested with restrictionendonucleases and cloned into vector and sequenced by Shanghai Sangon to confirmthe mutations were introduced. Allelic specific primers targeting wild-type andmutation-type templates were all designed with3’terminal phosphorothioatemodification. Two direction primer extension was carried out using mutationssensitive molecular switch mediated by high fidelity DNA polymerase in differentPCR systems and combined with agarose gel electrophoresis to detect the threemutations. Then multiplex primer extension was developed in the same system.Finally the mutations sensitive molecular switch combined with agarose gelelectrophoresis were used to discriminate the healthy clinical volunteers and patient’sDNA sample containing the three point mutations.Results: DNA sequencing confirmed the mutation-type templates harboring961delT, C1494T, and A1555G three mutations were successfully constructed byoverlap extension (SOE) PCR. The three mutations were detected respectively in different PCR systems. The results show that when wild-type plasmid template wasused, the mutations sensitive molecular switch can make the wild-type allele specificprimers extending, while the mutation-type allele specific primers cannot be extended.Conversely, with the using of mutation-type plasmid template, the allelic specificprimers targeting mutation-type can be extended and the wild-type allele specificprimers cannot be extended. Simultaneous detection of the three mutations in thesame system was established. Allelic specific primers perfectly matchingwild/mutation-type templates were extended, while no products were produced fromprimers mismatching mutation/wild-type templates. In the discrimination of the threepoint mutations in healthy clinical volunteers, the wild-type allele specific primersexactly matching the normal DNA sample can produce three specific products,mutation-type allele specific primers did not match with the normal DNA sample,there was no product appears.Conclusion: Mutation-type plasmid templates harboring the three hotspotmutations simultaneously associated with AAID can be constructed by the modifiedSOE PCR and used for genetic testing successfully. Mutations sensitive molecularswitch mediated by high fidelity DNA polymerase has high specificity and sensitivityin detecting single spot mutation associated with AAID. Mutations sensitivemolecular switch mediated by high fidelity DNA polymerase can be used in multipleprimer extension reaction to screen the three hot spot mutations closely related toAAID at the same time rapidly.