DNA separation electrophoresis gels and methods for their use

#The present invention generally relates to gels for separation mediums in electrophoretic systems. More particularly, the present invention involves improved separation medium gels and methods for their use in capillary electrophoresis based DNA separations.

#For decades electrophoretic separation techniques have been the method of choice for separating charged molecules and in particular for separating proteins. Early electrophoresis applications and many current applications involve applying electric fields across separation medium gels which are prepared in the form of slabs of varying size and thickness. Samples loaded at one end of the slab migrate across the slab under the influence of the electric field. When the sample's charged components have different electrokinetic mobilities they migrate at different rates and physically separate as a result of their differing electrokinetic mobilities. Traditionally, slabs which vary in size from several inches on each side to several feet are fabricated of a separation gel. Typically these gels are crosslinked polyacrylamide or other water swellable gel systems such as agarose or cellulose.

#In recent years capillary, electrophoresis (CE) techniques have become the electrophoretic separation method of choice for many biological researchers. Use of capillary electrophoresis for detection of DNA fragments is described by McGregor et al., "Detection of DNA Fragments Separated by Capillary Electrophoresis Based on Their Native Fluorescence Inside a Sheath Flow," Journal of Chromatography A, 680 (1984), 491-496; and Nishiwaka et al., "Separation of Long DNA Fragments by Capillary Gel Electrophoresis With Laser-Induced Fluorescence Detection," Electrophoresis, 1994, 15, 215-220. CE separations involve injecting samples into a buffer filled or gel filled capillary and generating the electric field across the capillary in order to cause sample components to electrophoretically migrate within the capillary. A variety of on-capillary column and off-capillary column detection techniques can be used to detect the components including uv, visible, fluorescence and electrochemical detection. CE offers many advantages over slab gel electrophoresis techniques. CE is available in fully automated systems which include automated injectors and data storage and analysis features and relatively easy to use detection systems. Additionally, CE separations are more rapid than slab gel separations and their separation mediums can be replaced after each analysis for a subsequent analytical run.

#These and other features, aspects, and advantages of the present invention will become better understood from the following description, appended claims, and accompanying drawing where:

#The FIGURE which schematically shows capillary electrophoresis system utilizing a gel separation medium according to the present invention.

#A gel according to this invention includes polyacrylamide and a dioxane in an effective resolution enhancing amount, preferably on the order of about 1 to about 5 grams polyacrylamide and about 5 to about 30 ml of a dioxane, per 100 ml of the gel.

#In the most preferred embodiments, the gel includes about 3 grams polyacrylamide, about 15 ml dioxane (1,4 dioxane and/or 1,3 dioxane), about 3.5 M urea, and about 100 mM tris-borate buffer, with the balance being water, to make a total gel volume of 100 ml.

#In accordance with another aspect of the present invention there is further provided electrophoresis separation apparatus including a separation channel and disposed within the separation channel a separation medium. The separation medium includes polyacrylamide and dioxane and preferentially further includes one or more biopolymer denaturants and an ionic buffer.