Detection Of ZNF644Mutations In High Myopia By Pfu High-fidelity DNA Polymerase With Double Phosphorothioate Modified Allele Specific Primers

Objective:Using the high fidelity pfu DNA polymerase with phosphorothioatemodified allele specific primers,established accurate and sensitive method fordetecting two ZNF644Mutations—I587V and3’UTR+592G>A in High Myopia.Bythis method we could detect and sceen ZNF644mutations and further verify thecorrelation of ZNF644gene and high myopia.Methods:Identified the I587V and3’UTR+592G>A positions sequences based onthe sequences of ZNF644gene in NCBI Gene database.Genomic DNA was extractedfrom normal blood.According to the sequences of known mutation region of I587Vand3’UTR+592G>A in ZNF644gene.The gene sequence primer was designed,andthen the primer extention reaction was performed.The PCR products was cloned intopGEM-T EASY vector.Two kinds of clones of ZNF644wild gene(I587V and3’UTR+592G>A) were obtained,and then obtained mutants by site-directedmutagenesis. Produced the recombinant plasmid as a template for research.3’-terminalprimer which was matched with mutation type or wild gene region was designed,and3’-terminal was modified with double phosphorothioate and linked to genemutation-sensitive molecular switch which was constructed by high-fidelity DNApolymerase.The mutation and wild plasmid template were administered by primerextention reaction and analyzed by gel-imagine system.Results:We have identified ZNF644gene sequence and the I587V and3’UTR+592G>A mutation location. Cloned the DNA fragments within the mutationpositions sequences, and obtained mutants by site-directed mutagenesis, constructedrecombinant plasmids containing the wild-type and mutant templets;When the mutantprimer was matched with the mutant-type plasmid template,the primer was extended;When the mutant primer was not matched with the wild-type plasmidtemplate,the primer was not extended.So the same, when the wide primer wasmatched with the wild-type plasmid template,the primer was extended;When the wildprimer was not matched with the mutant-type plasmid template,the primer was notextended.Conclusion: The high fidelity pfu DNA polymerase with phosphorothioatemodified allele specific primers could accurately and rapidly detect the mutation ofI587V and3’UTR+592G>A of ZNF644in high myopia.We could screened ZNF644mutations and further verify the correlation of ZNF644gene and high myopia by thismethold.