Electrophoresis Of Nucleic Acids

Amplified PCR products are visualized most often by running samples in an electrophoretic gel; staining the DNA within the gel with ethidium bromide, SYBR Green I, or another fluorescent dye with a high-affinity binding to DNA; and viewing the stained, separated PCR products under UV light. Nucleic acids are negatively charged and will run to the positive pole in an electric field. The gel matrix provides resistance to the movement of nucleic acids through it by virtue of the pore sizes within the gel, such that DNA fragments of smaller size will move through the matrix faster than those of a larger size. A standard molecular weight marker is typically run along with samples to enable the size of PCR products to be assigned during gel analysis. The analysis of the amplified products is based on the presence and pattern of DNA bands of various sizes contained in the gel matrix.

Agarose is the most popular medium for electrophoretic separation of medium-and large-sized nucleic acids. Agarose has a large working range, but poor resolution compared with polyacrylamide. Depending upon the agarose concentration used, nucleic acids between 0.1 and 70 kb in size can be separated. Polyacrylamide is the preferred matrix for separating proteins, single-stranded DNA fragments up to 2000 bases in length, or double-stranded DNA fragments of less than 1 kb. Polyacrylamide gels have excellent resolving power as they separate macromole-cules on the basis of configuration in addition to the more commonly exploited characteristics of size, charge, and G + C content. This shape-dependent mobility forms the basis of a suite of techniques that exploit inter- and intrastrand nucleotide interactions and can be used to screen amplified DNA rapidly for very fine-scale sequence differences. These techniques include single-strand conformation polymorphism (Dewit and Klatser, 1994), denaturing (or temperature) gradient gel electrophoresis (DGGE or TGGE) (Muyzer and Smalla, 1998), and heteroduplex mobility assays (Espejo and Romero, 1998). Because the electrophoretic mobility of nucleic acids using these techniques is highly sequence dependent, these techniques are often used in studies of genetic diversity.

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