高保真酶介导的突变敏感性分子开关用于三种常见遗传性疾病分子诊断的研究
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摘要
目的利用高保真聚合酶介导的突变敏感性分子开关，建立在同一PCR反应体系中对mtDNA12S rRNA基因A1555G和C1494T热点突变可以同时进行快速筛查的技术平台，指导AmAn的合理用药，从根本上全面预防AAID的发生。
方法mtDNA12S rRNA基因PCR产物克隆至pMD19-T载体，转化E.coli JM109感受态细胞，通过LB/Amp/IPTG/X-gal平板筛选白色克隆，载体通用引物对其进行菌液PCR扩增初步确定阳性克隆，并通过序列分析进行确证，得到mtDNA12S rRNA基因的野生模板质粒。反向PCR对其野生质粒模板实施体外mtDNA12S rRNA基因A1555G和C1494T定点突变，Dpn I内切酶降解甲基化的野生质粒DNA模板后，再次转化E.coli JM109感受态细胞，同前筛选阳性克隆，测序鉴定得到mtDNA12SrRNA基因C1494T和A1555G突变质粒模板。进一步设计与mtDNA12S rRNA基因A1555G和C1494T突变位点配对及三末端不配对的3’硫化修饰正向引物，在其下游设计一条公共反向引物，分别构成野生检测引物与突变检测引物，对mtDNA12SrRNA基因野生质粒模板和A1555G和C1494T突变质粒模板，进行高保真DNA聚合酶介导的双向引物延伸反应，利用凝胶成像系统对其PCR结果进行分析。并将其优化的PCR体系用于临床健康志愿者mtDNA12S rRNA基因C1494T和A1555G突变的筛查。
结果当突变检测引物与突变模板mtM配对时，引物被延伸，有PCR产物；与野生模板mt不配对时，引物却不能被延伸，无PCR产物。同样，野生检测引物只有与野生模板mt匹配时得以延伸，而与突变模板mtM不匹配时则不能延伸。突变敏感性分子开关对40例临床健康志愿者mtDNA12S rRNA基因C1494T和A1555G突变的筛查未发现C1494T和A1555G突变携带者。
结论高保真DNA聚合酶偶联硫化修饰引物构成的突变敏感性分子开关能够快速筛查mtDNA12S rRNA基因A1555G和C1494T突变，达到非此即彼的二元化效果，该分子开关在AAID基因筛查中具有较大的潜在应用价值。
目的利用高保真聚合酶介导的突变敏感性分子开关，建立在同一PCR反应体系中对NSHL患者GJB2基因235de1C、299-300delAT、176del16bp、35de1G突变，SLC26A4基因IVS7-2A>G、H723R突变，mtDNA12S rRNA基因A1555G、C1494T突变8个热点突变可以同时进行快速筛查的技术平台，达到指导个体化用药及为优生优育咨询提供保障的目的，造福耳聋患者。
方法GJB2基因PCR产物与SLC26A4基因重叠PCR拼接的DNA片段分别克隆至pMD19-T载体，转化E.coli JM109感受态细胞，通过LB/Amp/IPTG/X-gal平板筛选白色克隆，载体通用引物对其进行菌液PCR扩增初步确定阳性克隆，并通过序列分析进行确证，得到GJB2基因与SLC26A4基因的野生模板质粒。GJB2基因4条反向定点突变引物5’末端磷酸化后，与原始野生质粒模板GJB2进行退火、延伸、连接反应形成新的突变单链，再通过引物双向延伸反应获得突变双链DNA片段，突变双链DNA片段经EcoR I、Hind III双酶切、凝胶回收后克隆于目的载体中，转化E.coliJM109感受态细胞，LB/Amp/IPTG/X-gal平板筛选，随机挑选白色克隆用载体通用引物进行菌液PCR扩增和双酶切的初步鉴定后进行DNA测序，获得同时包含GJB2基因235de1C、299-300delAT、176del16bp、35de1G突变的突变质粒模板。先后通过反向PCR定点突变与重叠延伸产生特异性位点诱变方法对SLC26A4基因的野生模板质粒实施体外IVS7-2A>G、H723R定点突变后，再次转化E.coli JM109感受态细胞，同前筛选阳性克隆，测序挑选同时含有SLC26A4基因IVS7-2A>G、A2168G突变质粒模板。进一步设计分别与NSHL患者GJB2基因235de1C、299-300delAT、176del16bp、35de1G突变，SLC26A4基因IVS7-2A>G、H723R突变，mtDNA12S rRNA基因A1555G、C1494T突变位点配对及三末端不配对的3’硫化修饰引物，构成野生检测引物与突变检测引物，对GJB2基因、mtDNA12S rRNA基因与SLC26A4基因野生质粒模板和突变质粒模板，进行高保真DNA聚合酶介导的双向引物延伸反应，利用凝胶成像系统对其PCR结果进行分析。并将其优化的PCR体系用于临床NSHL8个特点突变的筛查。
结果GJB2与PDS分别为GJB2基因与SLC26A4基因的野生模板载体，其基因序列与Genebank来源一致。GJB2M与PDSM分别为GJB2与PDS的突变模板载体，GJB2M存在GJB2基因235de1C、299-300delAT、176del16bp和35de1G突变，PDSM存在SLC26A4基因IVS7-2A>G和A2168G突变。无论是突变位点的单靶检测，还是多靶同时检测，突变检测引物与突变模板配对时，特异性引物被延伸，有特异性PCR产物；与野生模板不配对时，特异性引物却不能被延伸，无特异性PCR产物。同样，特异性野生检测引物只有与野生模板匹配时得以特异性延伸，而与突变模板不匹配时则不能特异性延伸。突变敏感性分子开关对16例NSHL患者筛查GJB2基因235de1C、299-300del AT、176del16bp、35de1G突变，SLC26A4基因IVS7-2A>G、H723R突变，mtDNA12S rRNA基因C1494T、A1555G突变时，发现1例存在IVS7-2A>G突变，与测序结果一致。
结论高保真DNA聚合酶偶联硫化修饰引物构成的突变敏感性分子开关能够对NSHL相关的mtDNA12S rRNA基因C1494T和A1555G突变，GJB2基因35delG、176del16bp、235delC和299-300delAT突变，SLC26A4基因IVS7-2A>G和2168A>G8个热点突变同时快速筛查，达到非此即彼的二元化效果。
目的利用高保真聚合酶介导的突变敏感性分子开关，建立在同一PCR反应体系中对HCM患者bMHC基因Ala26Val、Arg663His、Arg723Gly、Gln893Lys、Ile736Thr、Glu924Lys突变，MYBPC3基因Ser236Gly、Glu258Lys、Arg346fs、Pro459fs、Lys814fs突变和TrpnI基因Arg145Trp热点突变可以同时进行快速筛查的技术平台，促进HCM的早期诊断与早期干预，评价HCM血缘亲属的发病风险，指导优生优育。
方法相应基因DNA片段的重叠PCR产物克隆至pMD19-T载体，转化E.coli JM109感受态细胞，通过LB/Amp/IPTG/X-gal平板筛选白色克隆，载体通用引物对其进行菌液PCR扩增初步确定阳性克隆，并通过序列分析进行确证，得到三个野生模板质粒。多条定点突变引物5’末端磷酸化后，与相应的原始野生质粒模板进行退火、延伸、连接反应形成新的突变单链，再通过引物双向延伸反应获得突变双链DNA片段，突变双链DNA片段经适当的限制性内切酶双酶切、凝胶回收后克隆于目的载体中，LB/Amp/IPTG/X-gal平板筛选，随机挑选白色克隆用载体通用引物进行菌液PCR扩增和双酶切的初步鉴定后进行DNA测序鉴定，得到相应的三个突变质粒模板。进一步设计与bMHC基因Ala26Val、Arg663His、Arg723Gly、Gln893Lys、Ile736Thr、Glu924Lys，MYBPC3基因Ser236Gly、Glu258Lys、Arg346fs、Pro459fs、Lys814fs和Trpn I基因Arg145Trp热点突变位点配对及三末端不配对的3’硫化修饰引物，分别构成野生检测引物与突变检测引物，对相应的野生质粒模板和突变质粒模板，进行高保真DNA聚合酶介导的双向引物延伸反应，利用凝胶成像系统对其PCR结果进行分析。并将其优化的PCR体系用于临床HCM患者12个特点突变的筛查。
结果MHC、MYB与MYT均为野生模板载体，其基因序列与Genebank来源一致。MHCM、MYBM与MYTM分别MHC、MYB与MYT载体的突变模板载体，MHCM存在bMHC基因Ala26Val、Arg663His、Arg723Gly、Gln893Lys、Ile736Thr、Glu924Lys突变，MYBM存在MYBPC3基因Ser236Gly、Glu258Lys、Arg346fs突变，MYTM存在MYBPC3基因Pro459fs、Lys814fs和Trpn I基因Arg145Trp突变。无论是突变位点的单靶检测，还是多靶同时检测，当突变检测引物与突变模板配对时，特异性引物被延伸，有特异性PCR产物；与野生模板不配对时，特异性引物却不能被延伸，无特异性PCR产物。同样，特异性野生检测引物只有与野生模板匹配时得以特异性延伸，而与突变模板不匹配时则不能特异性延伸。突变敏感性分子开关对8例临床HCM患者筛查未发现bMHC基因Ala26Val、Arg663His、Arg723Gly、Gln893Lys、Ile736Thr、Glu924Lys，MYBPC3基因Ser236Gly、Glu258Lys、Arg346fs、Pro459fs、Lys814fs和TrpnI基因Arg145Trp热点突变。
结论高保真DNA聚合酶偶联硫化修饰引物构成的突变敏感性分子开关能够同时对HCM相关的bMHC基因Ala26Val、Arg663His、Arg723Gly、Ile736Thr、Gln893Lys、Glu924Lys突变，MYBPC基因Ser236Gly、Glu258Lys、Arg346fs、 Pro459fs、Lys814fs突变和TrpnI基因Arg145Trp突变12个热点突变进行辨认。
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..
方法mtDNA12S rRNA基因PCR产物克隆至pMD19-T载体，转化E.coli JM109感受态细胞，通过LB/Amp/IPTG/X-gal平板筛选白色克隆，载体通用引物对其进行菌液PCR扩增初步确定阳性克隆，并通过序列分析进行确证，得到mtDNA12S rRNA基因的野生模板质粒。反向PCR对其野生质粒模板实施体外mtDNA12S rRNA基因A1555G和C1494T定点突变，Dpn I内切酶降解甲基化的野生质粒DNA模板后，再次转化E.coli JM109感受态细胞，同前筛选阳性克隆，测序鉴定得到mtDNA12SrRNA基因C1494T和A1555G突变质粒模板。进一步设计与mtDNA12S rRNA基因A1555G和C1494T突变位点配对及三末端不配对的3’硫化修饰正向引物，在其下游设计一条公共反向引物，分别构成野生检测引物与突变检测引物，对mtDNA12SrRNA基因野生质粒模板和A1555G和C1494T突变质粒模板，进行高保真DNA聚合酶介导的双向引物延伸反应，利用凝胶成像系统对其PCR结果进行分析。并将其优化的PCR体系用于临床健康志愿者mtDNA12S rRNA基因C1494T和A1555G突变的筛查。
结果当突变检测引物与突变模板mtM配对时，引物被延伸，有PCR产物；与野生模板mt不配对时，引物却不能被延伸，无PCR产物。同样，野生检测引物只有与野生模板mt匹配时得以延伸，而与突变模板mtM不匹配时则不能延伸。突变敏感性分子开关对40例临床健康志愿者mtDNA12S rRNA基因C1494T和A1555G突变的筛查未发现C1494T和A1555G突变携带者。
结论高保真DNA聚合酶偶联硫化修饰引物构成的突变敏感性分子开关能够快速筛查mtDNA12S rRNA基因A1555G和C1494T突变，达到非此即彼的二元化效果，该分子开关在AAID基因筛查中具有较大的潜在应用价值。
目的利用高保真聚合酶介导的突变敏感性分子开关，建立在同一PCR反应体系中对NSHL患者GJB2基因235de1C、299-300delAT、176del16bp、35de1G突变，SLC26A4基因IVS7-2A>G、H723R突变，mtDNA12S rRNA基因A1555G、C1494T突变8个热点突变可以同时进行快速筛查的技术平台，达到指导个体化用药及为优生优育咨询提供保障的目的，造福耳聋患者。
方法GJB2基因PCR产物与SLC26A4基因重叠PCR拼接的DNA片段分别克隆至pMD19-T载体，转化E.coli JM109感受态细胞，通过LB/Amp/IPTG/X-gal平板筛选白色克隆，载体通用引物对其进行菌液PCR扩增初步确定阳性克隆，并通过序列分析进行确证，得到GJB2基因与SLC26A4基因的野生模板质粒。GJB2基因4条反向定点突变引物5’末端磷酸化后，与原始野生质粒模板GJB2进行退火、延伸、连接反应形成新的突变单链，再通过引物双向延伸反应获得突变双链DNA片段，突变双链DNA片段经EcoR I、Hind III双酶切、凝胶回收后克隆于目的载体中，转化E.coliJM109感受态细胞，LB/Amp/IPTG/X-gal平板筛选，随机挑选白色克隆用载体通用引物进行菌液PCR扩增和双酶切的初步鉴定后进行DNA测序，获得同时包含GJB2基因235de1C、299-300delAT、176del16bp、35de1G突变的突变质粒模板。先后通过反向PCR定点突变与重叠延伸产生特异性位点诱变方法对SLC26A4基因的野生模板质粒实施体外IVS7-2A>G、H723R定点突变后，再次转化E.coli JM109感受态细胞，同前筛选阳性克隆，测序挑选同时含有SLC26A4基因IVS7-2A>G、A2168G突变质粒模板。进一步设计分别与NSHL患者GJB2基因235de1C、299-300delAT、176del16bp、35de1G突变，SLC26A4基因IVS7-2A>G、H723R突变，mtDNA12S rRNA基因A1555G、C1494T突变位点配对及三末端不配对的3’硫化修饰引物，构成野生检测引物与突变检测引物，对GJB2基因、mtDNA12S rRNA基因与SLC26A4基因野生质粒模板和突变质粒模板，进行高保真DNA聚合酶介导的双向引物延伸反应，利用凝胶成像系统对其PCR结果进行分析。并将其优化的PCR体系用于临床NSHL8个特点突变的筛查。
结果GJB2与PDS分别为GJB2基因与SLC26A4基因的野生模板载体，其基因序列与Genebank来源一致。GJB2M与PDSM分别为GJB2与PDS的突变模板载体，GJB2M存在GJB2基因235de1C、299-300delAT、176del16bp和35de1G突变，PDSM存在SLC26A4基因IVS7-2A>G和A2168G突变。无论是突变位点的单靶检测，还是多靶同时检测，突变检测引物与突变模板配对时，特异性引物被延伸，有特异性PCR产物；与野生模板不配对时，特异性引物却不能被延伸，无特异性PCR产物。同样，特异性野生检测引物只有与野生模板匹配时得以特异性延伸，而与突变模板不匹配时则不能特异性延伸。突变敏感性分子开关对16例NSHL患者筛查GJB2基因235de1C、299-300del AT、176del16bp、35de1G突变，SLC26A4基因IVS7-2A>G、H723R突变，mtDNA12S rRNA基因C1494T、A1555G突变时，发现1例存在IVS7-2A>G突变，与测序结果一致。
结论高保真DNA聚合酶偶联硫化修饰引物构成的突变敏感性分子开关能够对NSHL相关的mtDNA12S rRNA基因C1494T和A1555G突变，GJB2基因35delG、176del16bp、235delC和299-300delAT突变，SLC26A4基因IVS7-2A>G和2168A>G8个热点突变同时快速筛查，达到非此即彼的二元化效果。
目的利用高保真聚合酶介导的突变敏感性分子开关，建立在同一PCR反应体系中对HCM患者bMHC基因Ala26Val、Arg663His、Arg723Gly、Gln893Lys、Ile736Thr、Glu924Lys突变，MYBPC3基因Ser236Gly、Glu258Lys、Arg346fs、Pro459fs、Lys814fs突变和TrpnI基因Arg145Trp热点突变可以同时进行快速筛查的技术平台，促进HCM的早期诊断与早期干预，评价HCM血缘亲属的发病风险，指导优生优育。
方法相应基因DNA片段的重叠PCR产物克隆至pMD19-T载体，转化E.coli JM109感受态细胞，通过LB/Amp/IPTG/X-gal平板筛选白色克隆，载体通用引物对其进行菌液PCR扩增初步确定阳性克隆，并通过序列分析进行确证，得到三个野生模板质粒。多条定点突变引物5’末端磷酸化后，与相应的原始野生质粒模板进行退火、延伸、连接反应形成新的突变单链，再通过引物双向延伸反应获得突变双链DNA片段，突变双链DNA片段经适当的限制性内切酶双酶切、凝胶回收后克隆于目的载体中，LB/Amp/IPTG/X-gal平板筛选，随机挑选白色克隆用载体通用引物进行菌液PCR扩增和双酶切的初步鉴定后进行DNA测序鉴定，得到相应的三个突变质粒模板。进一步设计与bMHC基因Ala26Val、Arg663His、Arg723Gly、Gln893Lys、Ile736Thr、Glu924Lys，MYBPC3基因Ser236Gly、Glu258Lys、Arg346fs、Pro459fs、Lys814fs和Trpn I基因Arg145Trp热点突变位点配对及三末端不配对的3’硫化修饰引物，分别构成野生检测引物与突变检测引物，对相应的野生质粒模板和突变质粒模板，进行高保真DNA聚合酶介导的双向引物延伸反应，利用凝胶成像系统对其PCR结果进行分析。并将其优化的PCR体系用于临床HCM患者12个特点突变的筛查。
结果MHC、MYB与MYT均为野生模板载体，其基因序列与Genebank来源一致。MHCM、MYBM与MYTM分别MHC、MYB与MYT载体的突变模板载体，MHCM存在bMHC基因Ala26Val、Arg663His、Arg723Gly、Gln893Lys、Ile736Thr、Glu924Lys突变，MYBM存在MYBPC3基因Ser236Gly、Glu258Lys、Arg346fs突变，MYTM存在MYBPC3基因Pro459fs、Lys814fs和Trpn I基因Arg145Trp突变。无论是突变位点的单靶检测，还是多靶同时检测，当突变检测引物与突变模板配对时，特异性引物被延伸，有特异性PCR产物；与野生模板不配对时，特异性引物却不能被延伸，无特异性PCR产物。同样，特异性野生检测引物只有与野生模板匹配时得以特异性延伸，而与突变模板不匹配时则不能特异性延伸。突变敏感性分子开关对8例临床HCM患者筛查未发现bMHC基因Ala26Val、Arg663His、Arg723Gly、Gln893Lys、Ile736Thr、Glu924Lys，MYBPC3基因Ser236Gly、Glu258Lys、Arg346fs、Pro459fs、Lys814fs和TrpnI基因Arg145Trp热点突变。
结论高保真DNA聚合酶偶联硫化修饰引物构成的突变敏感性分子开关能够同时对HCM相关的bMHC基因Ala26Val、Arg663His、Arg723Gly、Ile736Thr、Gln893Lys、Glu924Lys突变，MYBPC基因Ser236Gly、Glu258Lys、Arg346fs、 Pro459fs、Lys814fs突变和TrpnI基因Arg145Trp突变12个热点突变进行辨认。
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..
引文
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[45]Chen I, Limb CJ, Ryugo DK. The effect of cochlear-implant-mediated electricalstimulation on spiral ganglion cells in congenitally deaf white cats. J Assoc ResOtolaryngol.2010Dec;11(4):587-603
[46]Yoshinaga-Itano C, Sedey AL, Coulter DK, et al. Language of early-andlater-identified children with hearing loss. Pediatrics.1998Nov;102(5):1161-71.
[1] Maron BJ, Maron MS, Wigle ED, B et al. The50-year history, controversy, andclinical implications of left ventricular outflow tract obstruction in hypertrophiccardiomyopathy: from idiopathic hypertrophic subaortic stenosis to hypertrophiccardiomyopathy[J]. J Am Coll Cardiol2009;54(3):191-200.
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[5]王志民,邹玉宝,宋雷,等.超声心动图检查调查8080例成人肥厚型心肌病患病率[J].中华心血管病杂志2004;12:1090-4.
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[1] Thompson DC, McPhillips H, Davis RL, et al. Universal newborn hearing screening:summary of evidence. JAMA.2001;286(16):2000-10.
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[1] Maron BJ, Maron MS, Wigle ED, B et al. The50-year history, controversy, andclinical implications of left ventricular outflow tract obstruction in hypertrophiccardiomyopathy: from idiopathic hypertrophic subaortic stenosis to hypertrophiccardiomyopathy[J]. J Am Coll Cardiol2009;54(3):191-200.
[2] Coats CJ, Hollman A. Hypertrophic cardiomyopathy: lessons from history[J]. Heart2008;94(10):1258-63.
[3] Taylor MR, Carniel E, Mestroni L. Familial hypertrophic cardiomyopathy: clinicalfeatures, molecular genetics and molecular genetic testing[J]. Expert Rev Mol Diagn2004;4(1):99-113.
[4] Maron BJ, Gardin JM, Flack JM, et al. Prevalence of hypertrophic cardiomyopathy ina general population of young adults. Echocardiographic analysis of4111subjects inthe CARDIA Study. Coronary Artery Risk Development in (Young) Adults[J].Circulation1995;92(4):785-9.
[5]王志民,邹玉宝,宋雷,等.超声心动图检查调查8080例成人肥厚型心肌病患病率[J].中华心血管病杂志2004;12:1090-4.
[6] Medeiros Pde T, Martinelli Filho M, Arteaga E, et al. Hypertrophic cardiomyopathy:the importance of arrhythmic events in patients at risk for sudden cardiac death[J]. ArqBras Cardiol2006;87(5):649-57.
[7] Greaves SC, Roche AH, Neutze JM, et al. Inheritance of hypertrophic cardiomyopathy:a cross sectional and M mode echocardiographic study of50families[J]. Br Heart J1987;58(3):259-66.
[8] Jarcho JA, McKenna W, Pare JA, et al. Mapping a gene for familial hypertrophiccardiomyopathy to chromosome14q1[J]. N Engl J Med1989;321(20):1372-8.
[9] Hengstenberg C, Schwartz K. Molecular genetics of familial hypertrophiccardiomyopathy[J]. J Mol Cell Cardiol1994;26(1):3-10.
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