You have just completed an experiment using SDS-PAGE to study the subunit struc-ture of ferritin (iron storage protein). Ferritin is composed of two types of subunits, H and L, in about equal quantities. You have included in your electrophoresis several standard proteins of known molecular weight. Draw the final stained gel in the form of Figure 6.7.
Protein MW
Ferritin 500,000
Subunit H 22,000
Subunit L 19,000
-lactalbumin
a 14,200
Trypsinogen 24,000
Egg albumin 45,000
Bovine serum albumin 65,000
1The Human Genome Project was a federal, government-sponsored program to sequence all DNA in human chromosomes. The project was completed in 2001.
FIGURE 6.8 An apparatus for horizontal slab gel electrophoresis of nucleic acids.
Courtesy of Hoefer, Inc. www.hoeferinc.
com.
prevent renaturing of the nucleic acid fragments during electrophoresis. For de-tection, nucleic acid chains for sequencing must be end-labeled with or a fluorescent tag. - and -labeled nucleic acids on gels are detected by autora-diography (see later). Nucleic acids end-labeled with fluorescent molecules are detected by fluorimeter scanning of the gels. Many researchers working on the large and expensive Human Genome Project1have generated huge amounts of DNA sequence data. Much of this information is stored in computer data banks for use by researchers around the world.
Agarose Gel Electrophoresis
The electrophoretic techniques discussed up to this point are useful for analyz-ing proteins and small fragments of nucleic acids up to 350,000 daltons (500 bp) in molecular size; however, the small pore sizes in the gel are not appropriate for analysis of large nucleic acid fragments or intact DNA molecules. The standard method used to characterize RNA and DNA in the range 200 to 50,000 base pairs (50 kilobases) is electrophoresis with agarose as the support medium.
Agarose,a product extracted from seaweed, is a linear polymer of galac-topyranose derivatives. Gels are prepared by dissolving agarose in warm elec-trophoresis buffer. After cooling the gel mixture to 50˚C, the agarose solution is poured between glass plates as described for polyacrylamide. Gels with less than 0.5% agarose are rather fragile and must be used in a horizontal arrangement (Figure 6.8). The sample to be separated is placed in a sample well made with a comb, and voltage is applied until separation is complete. Precast agarose gels of all shapes, sizes, and percent composition are commercially available.
Nucleic acids can be visualized on the slab gel after separation by soaking in a solution of ethidium bromide, a dye that displays enhanced fluorescence when intercalated between stacked nucleic acid bases. Ethidium bromide may be added directly to the agarose solution before gel formation. This method allows monitor-ing of nucleic acids durmonitor-ing electrophoresis. Irradiation of ethidium bromide–treated gels by UV light results in orange-red bands where nucleic acids are present. Nucleic acids may also be stained with the new, fluorescent SYBR dyes, which are less toxic
35S
32P
32P,35S,
than the mutagenic ethidium bromide. The newest, least toxic, and most sensitive stains for nucleic acids are GelRed and GelGreen from Biotium, Inc. (See Section C.) The mobility of nucleic acids in agarose gels is influenced by the agarose concentration and the molecular size and molecular conformation of the nucleic acid. Agarose concentrations of 0.3 to 2.0% are most effective for nucleic acid sep-aration (Table 6.3). Adding ethidium bromide to the gel can retard DNA mobility.
Like proteins, nucleic acids migrate at a rate that is inversely proportional to the logarithm of their molecular weights; hence, molecular weights can be estimated from electrophoresis results using standard nucleic acids or DNA fragments of known molecular weight. The DNA conformations most frequently encountered are superhelical circular (form I), nicked circular (form II), and linear (form III).
The small, compact, supercoiled form I molecules usually have the greatest mobility, followed by the rodlike, linear form III molecules. The extended, circu-lar form II molecules migrate more slowly. The relative electrophoretic mobility of the three forms of DNA, however, depends on experimental conditions such as agarose concentration and ionic strength.
The versatility of agarose gels is obvious when one reviews their many applications in nucleic acid analysis. The rapid advances in our understanding of nucleic acid structure and function in recent years are due primarily to the development of agarose gel electrophoresis as an analytical tool. Two of the many applications of agarose gel electrophoresis will be described here.
Analysis of DNA Fragments after Digestion by Restriction Endonucleases
Restriction endonucleases are enzymes that recognize a specific base sequence in double-stranded DNA and catalyze cleavage (hydrolysis of phosphodiester bonds) in or near that specific region (see Chapter 10, Section B, p. 297). Many viral, bacterial, or animal DNA molecules are substrates for the enzymes. When each type of DNA is treated with a restriction endonuclease, a specific number of DNA fragments is produced. The base sequence recognized by the enzyme occurs only a few times in any particular DNA molecule; therefore, the smaller the DNA molecule, the fewer specific cleavage sites there are. Viral or phage DNA, for example, is cleaved into about 50 fragments depending on the enzyme used, whereas larger bacterial or animal DNA may be cleaved into hundreds or thousands of fragments. Smaller DNA molecules, upon cleavage with a particular enzyme, will produce a limited set of fragments. It is unlikely that this set of fragments will be the same for any two
TABLE 6.3 Effective Range of Separation of DNA by Agarose Agarose (% w>v) Effective Range (kb)
0.3 5–50
0.5 2–25
0.7 0.8–10
1.2 0.4–5
1.5 0.2–3
2.0 0.1–2
1 2 3 4 5 6 7 8
FIGURE 6.9 Restriction patterns produced by agarose electrophoresis of DNA fragments after endonuclease action. DNA molecular weight ladders have been electrophoresed on a 1%
agarose gel and then stained with SYBR Green I nucleic acid gel stain. Lanes 1 and 8 contain HindIII-cut DNA; lanes 2 and 7, HaeIII-cut X174 RF DNA; lanes 3 and 6, 1 kilobase pair DNA ladder; lane 4, 100 base pair DNA ladder; lane 5, EcoR-I-cut pUC19 DNA mixed with PstI-cut X174 RF DNA.
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different DNA molecules, so the fragmentation pattern can be considered a “finger-print” of the DNA substrate. The restriction pattern is produced by electrophoresis of the cleavage reaction mixture through agarose gels, followed by staining with ethidium bromide or SYBR dyes (Figure 6.9). The separation of the fragments is based on molecular size, with large fragments remaining near the origin and smaller fragments migrating farther down the gel. In addition to characterization of DNA