Separation of DNA and RNA

Nucleic acids come in a very wide range of sizes, from several dozen base pairs to many millions. No single technique can be used to separate them all. Instead, researchers analyze the nucleic acid molecules using the overlapping electrophoretic techniques of polyacrylamide, agarose, and pulse-field gel electrophoresis. Each technique places DNA or RNA molecules in an electric field. Because the nucleic acid fragments contain negatively charged phosphate groups along the backbone of the DNA molecule, they move toward the positively charged anode. As with proteins, the migration rate of nucleic acids through a gel depends on their conformation, the buffer composition, the concentration of the gel support, and the applied voltage.

Agarose Gels. The techniques discussed so far are good for separating proteins and small nucleic acid fragments from 5 to 500 base pairs. The small pores of the polyacrylamide gels, however, are not appropriate for larger DNA fragments or intact DNA molecules such as plasmids. Gels made of agarose, a natural seaweed product, are used to characterize nucleic acids that are 200 to 500,000 base pairs long.

Agarose gels, which can be purchased commercially, are prepared by dissolving purified agarose in warm electrophoresis buffer, cooling the solution to 50 °C (122 °F), and then pouring it into a mold, where it turns into a gel. Just as with polyacrylamide, the concentration of agarose in a gel determines the size of its pores. A comb placed in the gel before it sets produces the wells necessary for loading nucleic acid samples.

Nucleic acid fragments that are to be separated by size must be in "linearized" form. Plasmids, for example, must have their circular structure cut open using restriction enzymes before they are run on the gel. Otherwise, their rate of migration will depend on how supercoiled they are and whether they are nicked, instead of on their size. Nucleic acids that are separated in a gel can be seen with ethidium bromide or other stains.

Pulse-Field Gel Electrophoresis. The conventional agarose gel elec-trophoresis described above separates nucleic acid fragments smaller than 50,000 base pairs (50 kilobase pairs). Pulse-field gel electrophoresis separates huge pieces of DNA that are between 200 and 3,000 kilobase pairs long. In this technique the electric field is not held constant during the separation. Instead, its direction and strength are repeatedly changed, with the molecules reorienting themselves every time the current changes. The molecules then slither like a snake through the gel matrix, in a process known as "reptation," with smaller fragments moving faster than larger ones. As the gel runs, it heats up and becomes more fluid, with the pulsing allowing

Was this article helpful?

0 0

Post a comment