Study On Detection Methods Of Gene Mutation And Their Clinical Applications

Recently, the study on genetic alteration and polymorphism of organisms' genome is drawing people's attention. Since traditional methods used for detection of genetic polymorphism is labor-intensive and time-consuming, an efficient and accurate platform suitable for sweeping detection of genetic alteration in clinical samples is becoming even more important.In this thesis, efficient and inexpensive sieving medium for capillary electrophoresis (CE)-short-chain linear polyacrylamide (LPA) and coating capillaries were prepared. Based on these, a platform suitable for CE detection of genetic alteration and polymorphism was developed on ABI capillary genetic analyzer. The migration behavior and separation mechanism of small DNA fragments were investigated. Results further proved the rationality of Ogston mechanism and traditional theory of polymer coils shrinking in semi-dilute solution.In order to realize the economization and customization of CE-DNA analysis, several kinds of CE techniques with homemade sieving matrix, including SNaPshot technique used for detection of known single nucleotide polymorphism of genome, single-strand conformational polymorphism (SSCP) and constant denaturant capillary electrophoresis (CDCE) used for detection of unknown sequence alteration of genome, were developed and applied to analysis of genetic alteration and polymorphism in clinical samples. The effect of various parameters on the separation has been investigated. Results demonstrate that under optimized conditions, homemade short-chain LPA has higher sieving ability and shorter analysis time than commercial matrices, and is more suitable for sweeping detection in clinical samples.In order to prove the feasibility and veracity of above CE techniques, genetic alteration and polymorphism of tumor tissue samples were detected, pathways and susceptibility of colorectal cancer development were discussed. Furthermore, 270pathogenic bacteria representing 34 species in 14 genera were identified with the multiple SSCP analysis and RFLP analysis of rRNA gene. Based on the machine code of peak patterns developed in this thesis, the identification of bacteria species becomes much easier, and can be applied to the eventual use in clinical laboratory instead of traditional method.