Study On Molecule Electrophoresis Methods And Its Setup

In molecular biology, medicine and clinical diagnosis, and genetic testing field, how to test the gene rapidly and effectively has become an important task in many new interdisciplines, such as molecular biology, optoelectronic technology and modern scientific instruments and so on.It is an important and indispensable step to separate the DNA and RNA fragments depending on their size by conventional slab gel electrophoresis (SGE) in genetic testing project. Therefore, SGE has been widely applied in polymerase chain reaction (PCR) products analysis, Genome sequencing, polymorphism analysis, genetic mutation, and restriction fragments analysis. Separation of the large DNA and RNA molecule can be more efficient for gene analysis, however, it is difficult to separate the large DNA and RNA fragments efficiently by traditional SGE and capillary electrophoresis (CE) in a short time.Concentrating on this problem, this doctoral dissertation has made a deep research on the separation of large DNA and RNA molecule with modern optics technology, pulse field electricity, and gel molecule electrophoresis technology. In order to resolve the large DNA and RNA molecules, we researched the separation of DNA and RNA sample by direct current CE and pulse field capillary electrophoresis (PFCE) and summarized the effective ways to separate the samples.Focusing on the research of DNA separation by direct current CE and PFCE, we have firstly researched the DNA (0.1-1.0kbp) separation by direct current CE in HEC polymer. We also investigated the influence of electric field strength, polymer concentration, effective capillary length and sample injection on the separation performance of DNA. Based on this, we have proposed the variable electricity for DNA separation by CE, and the simulation demonstrates that it can realize the fast and effective separation of short DNA molecules.In order to effectively resolve the large DNA fragments, we have made a full research of the separation of DNA (0.1-10.0kbp) by square-wave PFCE on the basis of variable electric CE. We analyzed the parameters that may influence the separation performance. We find that square wave pulse field capillary electrophoresis can selectively improve the separation of large DNA molecules by optimizing the modulation depth, while the resolution of median DNA molecule is deteriorated. In order to separate the sample with high resolution for each DNA fragment (0.1-10.0kbp), we proposed the inversion field capillary electrophoresis and suceessfully resolved?-DNA restricted by EcoT14 I endonuclease.On the basis of DNA separation by PFCE, we made a research of RNA (0.1-10.0knt) separation in HEC (250K) by PFCE. To our knowledge, this is the first time for RNA separation in pulse field conditions. We also investigated the effect of polymer concentration, modulation depth, and pulse frequency on the separation performance. Because of the similar structure of RNA and DNA, a comparison of migration behavior of RNA and DNA under different pulse field conditions was also provided. Furthermore, we have proposed the on-line PFCE for separation of RNA. It can realize denaturing and separating the large RNA molecule simultaneously in the capillary. We analyzed the parameters that may influence the separation performance. We also researched the effects of different denaturants on RNA mobility in the polymer.