Construction Of Standard Plasmid Of MtDNA⁴⁹⁷⁷ Deletion In Human Blood

Objective:Construction of standard plasmid of mitochondrial DNA ⁴⁹⁷⁷bp (mtDNA ⁴⁹⁷⁷ )deletion called common deletion,To develop a real time quantitative PCR method with Tapman probe to discuss the relationshiop between the mtDNA ⁴⁹⁷⁷ deletion and noise- induced hearing loss(NIHL)。Methods : Firstly , by using PCR technolgy amplify the fragment of the mitochondrial DNA ⁴⁹⁷⁷bp(mt DNA⁴⁹⁷⁷) deletion,consensus sequence named D-LOOP and house-keeping gene of human separately.Then purify the purpose fragments after agarose gel electrophoresis and gel extraction . Next, insert the purpose gene fragments into vector of PMD19-T respectively,get the recombinant vector into competence cells and choose positive cloning strains. Augment the positive cloning strains and extract the plasmid from the strains. Detect the recombinant vector by enzyme digest and gene sequence analysis. Afterward,Based on calculated molecular weight of the recombinant plasmid copy number ,by utilization a real-time quantitative PCR method with a series of concentration gradient standard plasmids, analyze the amplification curve and standard curve to assess the successfully constructed standard plasmids.Results: 1 .Successfully Using synthetized primer amplify the fragments of. mt DNA⁴⁹⁷⁷ deletion called CD,consensus sequence named D-LOOP and house-keeping gene GAPDH of human separately. 2. Successfully insert the gene fragments into vector of PMD19-T respectively . 3. Successfully constructed standard plasmids by assessment of the real-time quantitative PCR method. The standard plasmids'sequencings were confirmed correct The quantitative standard amplification curve showed that they had good linear correlation and could be used for could be for the quantification of other samples.Conclusion: Successfully constructed the standard plasmids of mt DNA⁴⁹⁷⁷ deletion,mitochondrial consensus sequence and house-keeping gene of human.This will provide a quantitative method to detect the mitochondrial DNA copy number and lay a foundation for further study of gene susceptibility in the noise-induced hearing loss .