Separation Of DNA Fragments With A Broad Range Of Molecular Weights By Capillary Electrophoresis With Sieving Matrix Of Poly(Ethylene Oxide)

Capillary electrophoresis has been concerned extensively in medicine and biology because of its high separative efficiency, rapidly analysis, small amount of sample, automation, and so on. It has been the most widely used technology in separation of charged biological macromolecules. DNA fragments with the similar charge-mass ratio are separated by capillary sieving electrophoresis. Capillary sieving electrophoresis contains capillary gel electrophoresis and non-gel sieving capillary electrophoresis. Samples are separated based on different molecular size. The uncross-linked and low-viscosity linear polymers are used as sieving matrices in capillary non-gel sieving electrophoresis. Non-gel sieving capillary electrophoresis has the following advantages: there are a lot of potential water-soluble polymers for development, and sieving matrices can be changed easily, and sieving matrices can be discharged from the capillary after electrophoresis. Sieving matrices can be washed in the column again, increasing the reproducibility. Sample will not remain in the column. Although its resolution is lower than capillary gel electrophoresis, it has long column- life, simply operation and well reproducibility. Therefore it has broad application prospects in separation of biological macromolecules, in particular the nucleic acids. The properties of sieving matrices impact DNA molecules directly in resolution, read length, reproducibility, injection time, injection pressure and capillary life, and so on. So researching the properties of sieving matrices is one of the most important works.Polyethylene oxide is a hydrophilic, electrically neutral linear polymer. It can automatically be coated in column and has longer life than the cellulose polymer, and is more stable than linear polyacrylamide. It can prevent the sample from adsorbing in column wall with low concentrations, so enhancing the separation efficiency and analytical reproducibility. Polyethylene oxide can serve as a molecular sieve to separate molecules or particles of different sizes with high concentrations.In my experiment, with poly ethylene oxide (PEO) as sieving matrix, DL 5000 DNA Markers(100, 250, 500, 750, 1000, 1500, 2000, 3000, 5000 bp) were taken as tested objects to study the effects of electric field intensity, the sieving matrix concentration,pH value, EB contents on separating double-chain DNA by using capillary electrophoresis and seek the optimum electrophoresis conditions. The optimum conditions for capillary electrophoresis was as 0.5% PEO, pH value of 8.0, EB contents of 3.0μg/mL, and voltage of 12.0 kV. The cloning gene of SDH and ETR1 were detected by this method. In brief, non-gel sieving capillary electrophoresis greatly improved the resolution and sensitivity for separation DNA fragments.