Water for Nucleic Acid Electrophoresis
Application Overview
Electrophoresis is the migration of a charged particle under the influence of an electric field. Positively charged particles migrate towards the cathode, and the negatively charged ones towards the anode. Their rate of migration depends on the strength of the field, on the net charge, size and shape of the particles (i.e., molecules) and also on the ionic strength, viscosity and temperature of the medium in which the molecules are moving. As an analytical tool, electrophoresis is simple, rapid and highly sensitive. Since many important biological molecules, such as nucleic acids and proteins, have ionizable groups, they exist as electrically charged species at any given pH. As such, electrophoresis is one of the most widely-used techniques in biochemistry and molecular biology.
Nucleic acids have a negative charge because of their phosphate backbone, and as such migrate toward the anode. Separation of nucleic acids by electrophoresis takes place within a support matrix or "gel", the most commonly used ones being agarose or polyacrylamide. Normally, the gel is cast in the shape of a thin slab, with wells for loading the sample. The gel is immersed within an electrophoresis buffer that provides ions to carry a current and some type of buffer to maintain the pH at a relatively constant value. The support matrix provides a means of separating molecules by size, in that they are porous gels. A porous gel may act as a sieve by retarding, or in some cases completely obstructing, the movement of large macromolecules while allowing smaller molecules to migrate freely.
Agarose is a polysaccharide extracted from seaweed. It is typically used at concentrations of 0.5 to 2%. The higher the agarose concentration, the "stiffer" the gel. Agarose gels have a large range of separation, but relatively low resolving power. By varying the concentration of agarose, fragments of DNA from about 200 to 50,000 bp can be separated using standard electrophoretic techniques. Polyacrylamide gels are cross-linked polymer of acrylamide. The length of the polymer chains is dictated by the concentration of acrylamide used, which is typically between 3.5 and 20%. Polyacrylamide gels have a rather small range of separation, but very high resolving power. In the case of DNA, polyacrylamide is used for separating fragments of less than about 500 bp. However, under appropriate conditions, fragments of DNA differing is length by a single base pair are easily resolved. In contrast to agarose, polyacrylamide gels are used extensively for separating and characterizing mixtures of proteins.
For the majority of DNA and RNA samples, electrophoretic separation is carried out in agarose gels. Since the charge per unit length in any given fragment of DNA or RNA is the same because of the phosphate backbone, all samples move towards the anode with the same mobility under an applied electric field. The separation in the agarose matrix takes place because it resists movement of the nucleic acids. The largest molecules will have the most difficulty passing through the gel pores, whereas the smallest molecules will move without much difficulty. As such, the mobility of nucleic acids during gel electrophoresis will depend on size, with the smallest molecules moving fastest.
Once the gel has been run, the nucleic acids in the gels need to be stained and visualized. The most commonly used staining reagent is the fluorescent dye ethidium bromide. This molecule intercalates with the nucleic acid molecule and can be viewed under UV light. The gel shows bands corresponding to different nucleic acid molecules populations with different molecular weight. Fragment size is usually reported in "nucleotides", "base pairs" or "kb" (for thousands of base pairs) depending upon whether single- or double-stranded DNA has been separated. Fragment size determination is typically done by comparison to commercially available DNA ladders containing linear DNA fragments of known length.
So far, the nucleic acids being separated by electrophoresis that have been discussed are in their native state. The electrophoresis condition in such a case is called non-denaturing. Denaturing conditions are used when DNA sequences are to be determined, or when the size of RNA is to be determined. DNA sequencing gels are run in the presence of denaturing agents such as urea and formamide, and since the DNA molecules being analyzed are shorter, acrylamide gels are used instead of agarose. Determining the size of RNA by gel electrophoresis requires denaturation of the RNA with denaturing agents such as formaldehyde, glyoxal and methylmercuric hydroxide.
To identify the presence of a specific nucleotide sequence in a gel, a technique called Southern blotting (if DNA is being analyzed) or Northern blotting (RNA is being analyzed) is used. In both cases, the DNA or RNA from the intact gel is transferred onto a piece of nitrocellulose or nylon membrane. The membrane is then exposed to a hybridization probe—a single DNA fragment with a specific sequence whose presence in the target DNA (or RNA) is to be determined. The probe DNA is labeled so that it can be detected, usually by incorporating radioactivity or tagging the molecule with a fluorescent or chromogenic dye.
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