Detection Of The ZNF644Mutation Of High Myopia By Mutation Sensitive Molecular Switch

Objective To detect the ZNF644c．725C＞T，C699Y（2238G＞A） mutation ofhigh myopia by the approach of sensitive molecular switch consisting of3′phosphorothioate modified allele specific primers and high-fidelityDNA polymerase.Methods The wild type and mutant type clones of the gene ZNF644wereobtained according to sequences of the mutations in the gene.3′-terminalprimers which were matched with wild or mutant type were designed andsynthesized. The3′-terminal of these primers were modified with doublesulfide phosphatethe, and linked to gene mutation-sensitive molecularswitch. The later was constructed by reaction catalyzed by high-fidelityDNA polymerase. The plasmid templates of the mutant and wild type geneswere then amplified by Polymerase Chain Reaction (PCR) in the presence ofthe primers. The PCR products were analyzed by gel-imagine system. Thereaction condition was optimized to establish protocol of detecting themutations of gene ZNF644by mutation sensitive molecular switch. Using theestablished protocol, we screened for the gene ZNF644c．725C＞T，C699Y（2238G＞A） mutations in12volunteers who are suffering high myopia.Results When the primer was fully matched with the template, the primerwas extended, leading to PCR products. In contrast, when the primer wasmismatched with the template, the primer could not be extended. Thereforethis study established the specificity of the protocol using the specificprimers that were modified at3′-terminal with double sulfidephosphatethe and linked to gene mutation-sensitive molecular switch, in combination with PCR catalyzed by high-fidelity DNA polymerase. We havescreened the gene ZNF644.mutations in12high myopia volunteers. Nopositive result was found in this population of the patients.Conclusions The detection protocol based on high fidelity PCR usingprimers that were3′-terminal modified with double sulfide phosphatetheand linked to gene mutation-sensitive molecular switch in combination withhigh-fidelity DNA polymerase can be used to accurately and rapidly screenthe c．725C＞T，C699Y （2238G＞A） mutations of gene ZNF644associatedwithy high myopia.