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polypeptide, and leads to low solubility in the deoxygenated form of hemoglobin. The DNA isolated from the white blood cells of patients is tested using the Mst II restriction endonuclease for digestion, followed by agarose gel electrophoresis and hybridization to a 32P-labeled DNA probe, where the probe sequence contains the entire human b-globin coding sequence.8 Individuals with sickle-cell anemia lack one of the Mst II cleavage sites, so that the DNA of these individuals will produce fewer fragments, and some normal fragment lengths will be missing. Figure 10.6 shows the autoradiogram of the separation patterns obtained by this method. Note that only the fragments that contain the b-globin coding sequence are visualized. In Figure 10.6, it can be seen that differences in the restriction fragment lengths occur in the 1.15 1.35 kilobase region of the separation pattern. After quantitative
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Figure 10.6. Detection of the sickle-cell anemia b-globin allele.8 [Reprinted, with permission, from G. H. Keller and M. M. Manak, DNA Probes , Macmillan (Stockton Press), New York, 1993. # Macmillan Publishers Ltd. 1993.]
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DNA FINGERPRINTING AND RESTRICTION FRAGMENT LENGTH POLYMORPHISM
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digestion of the DNA by the Mst II restriction enzyme, the normal (beta-A) DNA yields a 1.15 kilobase fragment. Because one of the restriction sites is absent on the sickle-cell gene (beta-S), this region of the DNA is not cleaved, and the resulting fragment is longer, at 1.35 kilobases. The patterns obtained for DNA from the mother and father both show the normal and the sickle-cell bands, while an affected child exhibits only the sickle-cell band at 1.35 kilobases. The results of this test clearly show that the fetus will have sickle-cell disease. 10.7. DNA FINGERPRINTING AND RESTRICTION FRAGMENT LENGTH POLYMORPHISM9 DNA ngerprinting methods are used in forensic labs for the identi cation of individuals, and in clinical labs for the diagnosis of genetic disease. These methods employ restriction enzymes that have been carefully selected by screening methods for their particular cleavage sites. Restriction enzymes cocktails are used in forensic methods to produce many fragments, so that sequence differences that normally occur between individuals produce many differences in fragment lengths. Diagnostic methods employ restriction enzymes that recognize a critical sequence where mutations of this sequence are known to be associated with the disease state. The term restriction fragment length polymorphism, abbreviated RFLP, refers to the variety of fragment lengths from DNA digestion with restriction endonucleases. The various lengths can result in such distinct electrophoretic separation patterns that they are like ngerprints because they are unique to an individual. Simple polymorphisms, such as that seen in Figure 10.6 with the Mst II site lacking in sicklecell patients, can be employed for diagnostics. Figure 10.7 illustrates the principles of RFLP methods. If the total number of fragments resulting from quantitative digestion is relatively small, the detection step may involve simple ethidium staining directly on the gel, with no Southern blot. However, if many fragments are produced, Southern blotting and detection via hybridization with labeled DNA probes are necessary. The probes that are chosen hybridize with many fragments, so that a signi cant
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Figure 10.7. Restriction fragment length polymorphism for DNA identi cation.
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Direct (monocloning) method Separate fragments over agarose gel
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Figure 10.8. The RFLP for paternity testing. Step 1 (top) is the isolation and cleavage of native DNA into restriction fragments; Step 2 (middle) is the agarose electrophoretic separation, which yields smears of indistinguishable bands; and Step 3 (bottom) shows the pattern obtained after Southern blotting, hybridization with 32P labeled probes, and autoradiography.
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portion of the separation pattern is visualized, and individual DNA differences are readily observed. Figure 10.8 illustrates DNA ngerprinting for paternity testing. The separation pattern resulting from the child s DNA can be seen to contain bands present in both the mother s and the father s DNA, while a new band in the child s DNA suggests a possible new mutation. The critical reagent in RFLP methods is the restriction enzyme (or cocktail of enzymes), which must produce very distinct fragment lengths for individual DNA samples. Diagnostic RFLP methods employ DNA that is readily available from the white cells of patient blood samples. Forensic methods, on the other hand, use DNA samples of unpredictable (usually low) quantity; furthermore, if the DNA sample is old and has been exposed to sunlight (UV radiation), the DNA may be present as cleaved fragments rather than intact genes. In such cases, the available DNA is ampli ed in concentration using the polymerase chain reaction (PCR).10 PCR technology has revolutionized DNA analysis, and has made such general forensic tests feasible.
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