Multiplex PCR Molecular Diagnosis And Mutation Detection Of P53、HBB Gene Research And Advance Research Of TALENs In Gene Therapy Of AIDs

Part Ⅰ The application research of multiplex PCR technology mediated by the "common primers"Objective To establish the PCR reaction system and condition mediated by "common primers" in order to put into use of the future establishment of multiplex PCR system.Methods Design and synthesize "common primers" referring to bacteriophage genomic sequences, and test the specificity and sensitivity of the PCR system associated "common primers" with "chimeric primers" using the human genome as template.Results The "common primers" are highly specific and sensitive, and the "chimeric primers" can dilute down to106copies without affecting amplification efficiency associated with "commmon primers".Conclusions The PCR reaction system mediated by "common primers" can reduce the "chimeric primers" concentration dramatically, and thus lay a foundation for the further establishment of multiplex PCR system. Part Ⅱ Detection of p53、HBB point mutation by mutation sensitive molecular "on/off" switchObjective To establish the system of p53、HBB point mutation detection using mutation sensitive molecular "on/off" switch.Methods We have obtained the p53&HBB wide and mutation type plasmids with the usage of PCR and restriction enzymes methods. Allelic specific primers targeting wild type and mutation type templates were designed with3’ terminal phosphorothioate modification. Two-directional primer extension was performed using Pfu polymerases. The results were analyzed through gel imaging system.Results Allelic specific primers with3’terminal phosphorothioate modification perfectly matching mutation type template were extended while no products were produced from primers targeting wide type template. Similarly, allelic specific primers with3’terminal phosphorothioate modification matching wide type were only extended when amplified with wide type as template.Conclusions The mutation sensitive molecular "on/off switch" mediated by high fidelity exo+polymerase is highly reliable in the diagnosis of p53、HBB point mutation. Part Ⅲ The advance research of the application of TALENs in AIDS gene therapyObjective To construct the TALENs adenovirus transfection vectors and establish the related detection system for recombinant TALENs efficiency.Methods The recombinant vectors were gained using PCR and double restriction enzymes digestion strategy and GFP protein was introduced to detect the efficiency of recombinant TALENs.Results The target genes have recombinated into pDC316vector directionally, and cell transfection results proved the high efficiency of recombinant TALENs.Conclusions We have constructed the TALENs into adenovirus transfection vector correctly, and thus lay a foundation for the further HIV infection research and gene therapy of AIDS.