Electrophoretic Separation of Nucleic Acids

Polyacrylamide is a cross-linked polymer of acrylamide. These gels are more difficult to prepare than agarose. Monomeric acrylamide (which is a known neurotoxin) is polymerized in the presence of free radicals to form polyacrylamide. The free radicals are provided by ammonium persulfate and stabilized by TEMED (/V/V/V/V -tetramethylethylenediamine). The chains of polyacrylamide are cross-linked by the addition of methylene-bisacrylamide to form a gel whose porosity is determined by the length of chains and the degree of cross-linking. The chain length is proportional to the acrylamide concentration usually between 3.5 and 20%. Cross-linking bis-acrylamide is usually added at the ratio 2 g bis/38 g acrylamide. 

Polyacrylamide gels are poured between two glass plates held apart by spacers of 0.4 to 1.0 mm, and sealed with tape. Most of the acrylamide 

Percentage Acrylamide (w/v) with BIS at 1 20 Effective Range for Separation of Linear DNA (bp) 

After separation by gel electrophoresis, the required band is sliced out of the ethidium-stained gel and can be visualized under an ultraviolet (UV) light. Care is taken to cut out as little of the gel as possible, using a clean, sharp razor blade. The gel slice containing the DNA is then subjected to any of the following isolation techniques. 

Binding and Elution from Glass or Silica Particles 

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Capillary electrophoretic separation of nucleic acids has been reviewed (Cohen et al., 1987a Kuhr, 1990 Gebauer and Thormann, 1991). In nucleotide and nucleoside analysis, MEKC has been the method of choice, using SDS (Row et al. 1987 Cohen et al., 1987b Kasper et al., 1988), dodecyltrimethylammonium bromide, or hexadecyltriethylammonium bromide (Liu et al., 1989). Other applications concerning chemically modified nucleotides, nucleosides, and nucleobases can be found in papers by Lecoq et al. (1991) and Thormann et al. (1992). 

Consideration will be given to techniques requiring simple equipment that is inexpensive and readily available in most biomolecular science laboratories, as well as to the more sophisticated procedures that require highly expensive equipment that is available in only a few relatively specialized departments. Although electrophoretic analysis of nucleic acids also continues to be of considerable importance in clinical studies, this review will be concerned mainly with proteins, electrophoresis of nucleic acids having been considered in a separate article. 

We hope that this brief review has given the reader a general feeling of the development and application of CE in the separation of nucleic acids. With the advent of capillary array electrophoresis and microchip electrophoresis, as well as remarkable improvements in separation matrices, CE has become a standardized and cost-effective technique in the separation of nucleic acids. Novel thermo-responsive polymer solutions combine the merits of different monomers, and offer the possibility to fine-tune the desirable properties of polymer molecular architecture and chemical composition. Artificial entropic trapping systems obviate the use of viscous polymer solutions, and even offer fast, unattended, miniaturized, and multiplexed platforms. Optimizing the geometry of these electrophoretic systems to both increase the separation and reduce the diffusion (band broadening) is the main topic for future research. 

The initial characterizations of the various fusion proteins (Subheading 3.1.1, step 7), as well as more detailed biochemical assays (Subheading 3.3) make extensive use of electrophoretic separations and immunoblotting. Protocols used in the authors laboratories for these various techniques (SDS-polyacrylamide gel electrophoresis, native acrylamide gel electrophoresis, and separation of nucleic acid on acrylamide gels containing urea) are described in this section. 

SDS-Polyacrylamide Gel Electrophoresis Because many proteins or nucleic acids that differ In size and shape have nearly identical charge mass ratios, electrophoresis of these macromolecules in solution results in little or no separation of molecules of different lengths. However, successful separation of proteins and nucleic acids can be accomplished by electrophoresis In various gels (semisolid suspensions in water) rather than in a liquid solution. Electrophoretic separation of proteins is most commonly performed in polyacrylamide gels. When a mixture of proteins is applied to a gel and an electric current is applied, smaller proteins migrate faster through the gel than do larger proteins. 

Separations by electrophoresis depend upon differences in rates of migration of the components in a mixture in an applied electric field. Provided the electric field is removed before ions in the sample mixture reach the electrodes, the components may be separated according to their electrophoretic mobility. Electrophoresis is thus an incomplete form of electrolysis. Electrophoresis is especially useful for analysis and separation of amino acids, peptides, proteins, nucleotides, nucleic acids and carbohydrates. 

With CZE as described in section 3.3.2, it is often impossible to separate different nucleic acids from each other because they have a similar charge to size ratio and, thus, similar electrophoretic mobilities (equation 3.5). The same is true for SDS denatured proteins. Introducing a gel into the capillary, leads to an additional molecular sieving effect. Large analytes are retained more than smaller ones, enabling separation of analytes with similar mobilities. 

Kim, Y. Morris, M.D. Rapid pulsed field capillary electrophoretic separation of megabase nucleic acids. Anal. Chem. 1995,67 (5), 784-786. 

In the assay described here, no attempt is made to recover and purify the products of the RNase reactions. The traces of organic solvents that carry through into the aqueous phase of the reactions is minimal and does not interfere with the electrophoretic separation of the nucleic acids. However, the buffer components will affect electrophoretic behavior thus, it is very important to use equal volumes of sample, all of which have identical buffer compositions. 

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