Gene location

OFAGE, CHEF, various blots

Isolation and Preparation of DNA

DNA from most cellular organisms can be isolated through the disruption of cell membranes and/or walls by using lytic enzymes or other physicochemical forces (osmotic pressure, shear forces, and ultrasound). DNA of high (chromosomal) and low molecular weights (from plasmids, mitochondria, and chloroplasts) can be extracted. Because cellular lysates are rich in DNA-degrading enzymes, such reactions are performed in the presence of chelating agents or cooler temperature. DNA is then purified from proteins and other cellular constituents by extraction with a mixture of phenol-chloroform and is recovered by ethyl alcohol or isopropanol precipitation. To purify further, high-speed dye (ethydium bromide or bisbenzimide) buoyant density ultracentrifugation is performed and to characterize the plasmid or other types of DNA molecules, agarose gel electrophoresis is employed (Fig. 12). Although DNA isolated by this method is of high quality, the procedure is a lengthy one requiring expensive equipment. Nowadays, commercial DNA purification kits that utilize silica-based resins and anion exchange have become available. These kits allow for the isolation of DNA in purity equivalent or superior to that obtained by two successive rounds of cesium chloride gradient centrifugation (34). Such plasmid DNA is now routinely used in such applications as transfection, microinjection, automated and manual sequencing, restriction analysis and in vitro transcription.

In electrophoresis, DNA molecules migrate according to their molecular weight from the negative to the positive electrode; that is, the smaller molecules move further from the origin. If such DNAs are cut by any of some 600 restriction endonucleases, enzymes that cleave phosphodies-ter linkages at specific sequences within DNA, the resultant fragments can be separated in an electrophoretic

Figure 12. Separation of chromosomal and plasmid DNA from the bacterium Bacillus thuringiensis HD-1 by agarose gel electrophoresis (a) and chromosomal and mitochondrial DNA from the fungus Beauveria bassiana by cesium chloride bisbenzimide gradient centrifugation (b).

separation gel, according to their sizes (Fig. 13). DNA fragments after electrophoresis can be transferred permanently by blotting onto specific membranes. Often the electrophoretic separation of DNA followed by the ethidium bromide staining is used for visualization of DNA molecules, although the same can be achieved by electron microscopic analysis for size (Figs. 4 and 11) or heteroduplex analysis. Table 2 lists most commonly used enzymes for cell lysis, DNA extraction, and subsequent rDNA construction. While all DNA-sequencing protocols are based on the Sanger's chemical method (Fig. 14), other subsets of methodology by enzymatic (Klenow polymerase, Taq polymerase, and polymerase chain reaction, or PCR) systems are also used. Sequencing tasks are now automated and commercially available both for synthesis and sequencing (33).

Polymerase Chain Reaction

The PCR developed by Cetus (Emeryville, California) scientists in 1985 (35) is a powerful in vitro method for amplifying a segment of DNA that lies between two regions of known sequence, defined by a set of primers. The basic steps of PCR are denaturation of the DNA, annealing the primers to complementary sequences, and extension of the annealed primer with a thermostable DNA (eg, taq) polymerase. Together these steps represent one cycle of amplification.

As illustrated in Figure 15 the double-stranded DNA template is first denatured by heating in the presence of a

Hind HI Bglir C la I

Figure 13. Restriction endonuclease digestion of bacteriophage lambda DNA into fragments. The names of enzymes are indicated on the top of each track.

large molar excess of two specific oligonucleotides and four dNTPs. The reaction is then allowed to cool to a temperature that allows for the annealing of pairs of oligonucleotides to their target sequences. Once annealing has occurred DNA polymerase mediates the 5' to 3' extension of the primer-template complex. These three steps are repeated several times and the major product of this reaction

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