Study On The Technology Of Correcting Point Mutation By Modified Single-stranded Oligonucleotides

Objective: The technique that correcting the point mutation by specially designed oligonucleotides(ONs) is a latest molecular biological technique developed in recent years. With this technique, we can correct or introduce a assigned point mutation in any site of genes, which sequence already known. Contrast to the traditional gene recombination method, this technique hasn't any influence on the construction of genome itself and doesn't produce immunoreaction and random integration caused by virus or plasmid vectors. Some genetic diseases, such as Duchenne muscular dystrophy (DMD), are partly caused by a given point mutation. In this study, we aim to establish a new empirical method for studying oligonucleotide-mediated gene repair of point mutation. We tested the ability of the different designed single-stranded oligonucleotides (ODNs) to correct the point mutation, determined the better structure and the optimal concentration of ODN. In vivo studies,we had tried to correct the point mutation of dystrophin gene by specially designed single-stranded oligonucleotides and provided some experimental data for this technique to be ultimately used in gene therapy of DMD or other diseases caused by point mutation.Methods: This study was divided into three parts. In the first part, basing on Ames test system, we had tried to establish a new empirical method in which we used ODN to repair the point mutation in the gene of histidine operon of TA102. First, we tested ODN's ability to correct the point mutation by Ames test; then explored the correcting state when ODN mixed in bacterium which was in the period of rapid growth and,at last,studied ODN's ability to correct the point mutations when it mixed in competence bacterium which prepared by CaCl2. In the second part, several different concentrations of ODN and different structures of ODNs were performed to correct the point mutation in the gene of histidine operon of TA102.The different ODNs including 31,45,60,75bp length of template strands and 31bp length of coding strands were used. In vivo studies we administrated low does and high does ODN to DMDmdx mouse by intramuscular injection, or pretreated with hyaluronidase and did electroporation in vivo in addition. We tried to detect the corrected gene by amplification refractory mutation system (ARMS) assay.Conclusion: ODN can repair the point mutation in the gene of histidine operon of TA102 and the empirical method which baseing on Ames test system used for studying gene repair of point mutation by ODN is available. In this new system, strand bias is exist when using ODN to repair the point mutation in the gene of histidine operon of TA102; the 75bp length of template strand has a better repair efficient relatively, and 5ng/uL is the best concentration. But in vivo studies,what we used methods cannot correct the point mutation of dystrophin gene in vivo, or the repair efficiency was too low to be detect by ARMS assay.