Establishment Of Three Common Genetic Diseases Molecular Diagnostic Assays By Mutation Sensitive Molecular Switch

Objective To apply the “on/off” switch consisting of3’ phosphorothioate modified allelespecific primers and exo+polymerase in single base discrimination of A1555G andC1494T mutations in the highly conserved site of the mitochondrial DNA (mtDNA)12SrRNA. The two point mutations are the hotspot mutations associated with eitheraminoglycoside antibiotics induced deafness (AAID) or inherited nonsyndromic deafness.Methods The PCR products of mtDNA12S rRNA gene were inserted into the pMD19-Tvector for transformation into E.coli JM109competent cells for preparing wild typemtDNA vector. Inverse PCR was carried out for mtDNA12S rRNA gene C1494T andA1555G mutagenesis and DpnI endonuclease degradating methylated wild type mtDNAvector in inverse PCR products was carried out to construct the mutation type mtDNAvector. These constructed vectors were confirmed by DNA sequencing. Allelic specificprimers targeting wild type and mutation type templates were designed with3’ terminalphosphorothioate modification. Two-directional primer extension was performed using Pfupolymerases.Results The mtDNA12S rRNA gene wild type and mutation type plasmid vector werenamed mt and mtM vector separately. mt vector sequence was consistent with GeneBankDNA sequence, mtM vector contained mtDNA12S rRNA gene C1494T and A1555G mutations. Whether single PCR or multiplex PCR amplification by exo+polymerase, allelicspecific primers perfectly matching wild type allele were extended while no products wereproduced from primers targeting point-mutated AAID-related allele. Similarly, allelicspecific primers perfectly matching point-mutated AAID-related mutation type allele wereextended and no products were yielded from primers targeting wild type allele. No specificproduct was observed in the primer extension reaction mediated by on/off switch inscreening the mtDNA12S rRNA gene harboring either C1494T or A1555G mutation in40healthy volunteers tested.Conclusions These data suggest that the “off-switch” mediated by exo+polymerase ishighly reliable in the diagnosis of monogenic diseases and the novel “on/off” switch hasenormous applications in systematic and extended screening mtDNA12S rRNA geneA1555G and C1494T mutations. The established assay can be widely used not only forhearing loss patients but also for normal subjects before the use of aminoglycosideantibiotics. Objective To apply the “on/off” switch consisting of3’ phosphorothioate modified allelespecific primers and exo+polymerase in single base discrimination of Gap Junction Protein2(GJB2) gene235de1C,299-300delAT,176del16bp,35de1G mutations, solute carrierfamily26member4(SLC26A4) gene IVS7-2A>G, H723R mutations, mtDNA12S rRNAA1555G, C1494T mutations. These eight point mutations are the hotspot mutationsassociated with inherited nonsyndromic hearing loss (NSHL).Methods GJB2gene PCR products and Overlapping PCR-generated SLC26A4geneFragment were separately inserted into the pMD19-T vector for transformation into E.coli JM109competent cells for preparing wild type GJB2gene and SLC26A4gene plasmidvector. After5’ terminal phosphorylation of four reverse mutagenesis primers, Annealing,extension, ligation with GJB2gene wild type plasmid vector template were executed toform a new mutant single chain, and then through the primer extension reaction to obtainmutant double-stranded DNA fragment, which was cloned into the destination vector fortransformation into E.coli JM109competent cells for preparing mutation type GJB2geneplasmid vector after EcoR I, Hind III restriction enzyme digestion. Inverse PCR-generatedand overlapping PCR-generated specific locus mutagenesis methods were carried out forSLC26A4gene IVS7-2A>G and H723R mutagenesis and the mutation type SLC26A4gene vector construction. These constructed vectors were all confirmed by DNAsequencing. Allelic specific primers targeting wild type and mutation type templates weredesigned with3’ terminal phosphorothioate modification. Two-directional primer extensionwas performed using polymerases with3’ exonuclease activity.Results The GJB2vector and PDS vector were GJB2gene and SLC26A4gene wild typeplasmid vector respectively, the sequence were consistent with GeneBank DNA sequence.The GJB2M vector and PDSM vector were GJB2gene and SLC26A4gene mutation typeplasmid vector respectively, GJB2M vector contains GJB2gene235de1C,299-300delAT,176del16bp and35de1G mutations, and PDSM vector contains SLC26A4geneIVS7-2A>G and A2168G mutations. Whether single PCR or multiplex PCR amplificationby exo+polymerase, allelic specific primers perfectly matching wild type allele wereextended while no specific products were produced from primers targeting point-mutateddeafness-related allele. Similarly, allelic specific primers perfectly matching point-mutateddeafness-related mutation type allele were extended and no specific products were yieldedfrom primers targeting wild type allele. In screening DNA samples from16cases of NSHL,one IVS7-2A>G mutation was identified, consistent with DNA sequencing.Conclusions These data suggest that the “off-switch” mediated by exo+polymerase ismore reliable in the identification of NSHL hotspot mutation sites and the “on/off”mutation sensitive molecular switch has potential enormous application in the diagnosis of monogenic diseases. Objective To apply the “on/off” switch consisting of3’ phosphorothioate modified allelespecific primers and exo+polymerase in single base discrimination of beta-cardiac myosinheavy chain (bMHC) gene Ala26Val, Arg663His, Arg723Gly, Gln893Lys, Ile736Thr,Glu924Lys, cardiac myosin-binding protein C (MYBPC) gene Ser236Gly, Glu258Lys,Arg346fs, Pro459fs, Lys814fs, and cardiac troponin I (TrpnI) gene Arg145Trp mutations.These twelve point mutations are the hotspot mutations associated with hypertrophiccardiomyopathy (HCM).Methods Overlapping PCR-generated gene fragments were separately inserted into thepMD19-T vector for transformation into E.coli JM109competent cells for preparing wildtype gene plasmid vector. After5’ terminal phosphorylation of mutagenesis primers,Annealing, extension, ligation with wild type plasmid vector template were executed toform a new mutant single chain, and then through the primer extension reaction to obtainmutant double-stranded DNA fragment, which was cloned into the destination vector fortransformation into E.coli JM109competent cells for preparing mutation type plasmidvector after Hind III, EcoR I or BamH I restriction enzyme digestion. These constructedvectors were all confirmed by DNA sequencing. Allelic specific primers targeting wildtype and mutation type templates were designed with3’ terminal phosphorothioatemodification. Two-directional primer extension was performed using polymerases with3’exonuclease activity.Results MHC, MYB and MYT vector were wild type plasmid vectors. MHC vectorcontains bMHC gene3,18,20,23exon DNA fragments, MYB vector contains MYBPC gene7,13exon DNA fragments, MYT vector contains MYBPC gene16,26exon andTrpnI gene7exon DNA fragments. Their sequence were consistent with GeneBank DNAsequence. MHCM, MYBM and MYTM vector were MHC, MYB, MYT vector mutationtype plasmid vector respectively, MHCM vector contains bMHC gene Ala26Val,Arg663His, Arg723Gly, Gln893Lys, Ile736Thr and Glu924Lys mutations. MYBM vectorcontains MYBPC gene Ser236Gly, Glu258Lys and Arg346fs mutations. MYTM vectorcontains MYBPC gene Pro459fs, Lys814fs and TrpnI gene Arg145Trp mutations. Whethersingle PCR or multiplex PCR amplification by exo+polymerase, allelic specific primersperfectly matching wild type allele were extended while no specific products wereproduced from primers targeting point-mutated HCM-related allele. Similarly, allelicspecific primers perfectly matching point-mutated HCM-related mutation type allele wereextended and no specific products were yielded from primers targeting wild type allele. Nospecific product was observed in the primer extension reaction mediated by on/off switchin screening the HCM hotspot mutations in8HCM patients tested.Conclusions These data suggest that the “off-switch” mediated by exo+polymerase ismore reliable in the identification of HCM hotspot mutation sites and the “on/off” mutationsensitive molecular switch has potential enormous application in the diagnosis ofmonogenic diseases..