Restriction Endonuclease and Restriction Maps

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CLINICAL CORRELATION 18.1 Polymerase Chain Reaction and Screening for Human Immunodeficiency Virus
Use of the polymerase chain reaction (PCR) to amplify minute quantities of DNA has revolutionized the ability to detect and analyze DNA species. With PCR it is possible to synthesize sufficient DNA for analysis. Conventional methods for detection and identification of the human immunodeficiency virus (HIV), such as Southern blot–DNA hybridization and antigen analysis, are labor intensive, expensive, and have low sensitivity. An infected individual, with no sign of AIDS (acquired immunodeficiency syndrome), may test false negative for HIV by these procedures. Early detection of HIV infections in these individuals is crucial to initiate treatment and/or monitor the progression of their disease. In addition, a sensitive method is required to be certain that contributed blood from donors does not contain HIV. PCR amplification of potential HIV DNA sequences within DNA isolated from an individual's white blood cells permits the identification of viral infections prior to the presence of antibodies, the so­called seronegative state. Current methodologies are too costly to apply this testing to large­scale screening of donor blood samples. PCR can also be used to increase the sensitivity to detect and characterize DNA sequences of any other human infectious pathogen.
Kwok, S., and Sninsky, J. J. Application of PCR to the detection of human infectious diseases. In: H. A. Erlich (Ed.), PCR Technology. New York: Stockton Press, 1989, p. 235.
termed Taq DNA polymerase isolated from Thermus aquaticus, is now employed, obviating the need for fresh polymerase after each cycle. This has permitted the automation of PCR with each DNA molecule capable of being amplified one million­fold.
When the DNA to be amplified is present in very low concentrations relative to the total DNA in the sample, it is possible to amplify the DNA region of interest along with other spurious sequences. In this situation the specificity of the amplification reaction can be enhanced by nested PCR. After conducting the first PCR with one set of primers for 10–20 cycles, a small aliquot is removed for a second PCR. However, the second PCR is conducted with a new set of primers that are complementary to the template DNA just downstream of the first set of primers, or ''nested" between the original set of primers. This process amplifies the DNA region of interest twice with a greatly enhanced specificity.
PCR has many applications including gene diagnosis, forensic investigations where only a drop of dried blood or a single hair is available, and evolutionary studies with preserved biological material. Use of PCR for screening for human immunodeficiency virus is presented in Clin. Corr. 18.1.
18.3— Restriction Endonuclease and Restriction Maps
Restriction Endonucleases Permit Selective Hydrolysis of DNA to Generate Restriction Maps
Nature possesses a diverse set of tools, the restriction endonucleases, capable of selectively dissecting DNA molecules of all sizes and origin into smaller fragments. These enzymes confer some protection on bacteria against invading viruses, that is, bacteriophage. The bacterial DNA sequences normally recognized by the restriction endonuclease may be protected from cleavage in the host cell by methylation of bases within the enzyme recognized palindrome while the unmethylated viral DNA is recognized as foreign and is hydrolyzed. Numerous Type II restriction endonucleases, with differing sequence specificities, have been identified and purified; many are now commercially available (see p. 609 for discussion of restriction endonuclease activities).
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Restriction endonuclease permits construction of a new type of genetic map, the restriction map, in which the site of enzyme cleavage within the DNA is identified. Purified DNA species that contain restriction endonuclease sequences are subjected to restriction endonuclease cleavage. By regulating the time of exposure of the purified DNA molecules to restriction endonuclease cleavage, a population of DNA fragments that are partially to fully hydrolyzed can be generated. Separation of these enzyme­generated fragments by agarose gel electrophoresis allows for the construction of restriction maps; an example of this procedure with circular DNA is presented in Figure 18.2. Analysis of a DNA completely hydrolyzed by a restriction endonuclease establishes how many sites the restriction endonuclease recognizes within the molecule and what size fragments are generated. The size distribution of composite fragments generated by the partial enzymatic cleavage of the DNA molecules demonstrates linkage of all potential fragments. The sequential use of different restriction endonucleases has permitted a detailed restriction map of numerous circular DNA species including bacterial plasmids, viruses, and mitochondrial DNA. The method is also equally amenable to linear DNA fragments that have been purified to homogeneity.
Restriction Maps Permit the Routine Preparation of Defined Segments of DNA
Restriction maps may yield little information as to the genes or regulatory elements within the various DNA fragments. They have been used to demonstrate sequence diversity of organelle DNA, such as mitochondrial DNA, within species (see Clin. Corr. 18.2). Restriction maps can also be used to detect deletion mutations where a defined DNA fragment from the parental strain
Figure 18.2 Restriction endonuclease mapping of DNA. Purified DNA is subjected to restriction endonuclease digestion for varying times, which generates partially to fully cleaved DNA fragments. The DNA fragments are separated by agarose gel electrophoresis and stained with ethidium bromide. The DNA bands are visualized with a UV light source and photographed. The size of the DNA fragments is determined by the relative migration through the gel as compared to co­electrophoresed DNA standards. The relative arrangement of each fragment within the DNA molecule can be deduced from the size of the incompletely hydrolyzed fragments.