EXPANDINO THE FRONTIERS OF SCIENCE 1:
Keith B. Belton
American Chemical Society
Continental drift. Evolution. The biochemistry of healing. What do these disparate topics have in common? Recent discoveries based on information provided through modem techniques in DNA analysis have shed new light on each of these subjects. With such techniques, forensic scientists can now positively identify a criminal from trace amounts of DNA left a t a crime scene; medical researchers can locate the genetic defects responsible for many inherited diseases; and doctors can diagnose certain diseases, including some cancers, much more rapidly than ever before. Two of these techniques, DNA fingerprinting and DNA amplification, are generating much of the excitement in DNA analysis. DNA lingerprinting A DNA fingerprint is as unique to a particular individual as a conventional fingerprint. Except for identical twins, no two people have DNA that is exactly alike. Because of this, restriction en. zymes, which are used to cut DNA a t specific nucleotide sequences, will cut each individual’s DNA into differentsized fragments. Alee Jeffrey8 and his colleagues at the University of Leicester ( 1 ) have discovered certain groups of repeating nucleotide sequences in DNA. These regions, which they term minisatellites, differ among individuals in the number of repeating core sequences. Digestion of DNA with restriction enzymes results in DNA fragments of different lengths attributable in part to the differences in the number of core sequences in the minisatellite
regions. These fragments can be separated by electrophoresis, and then radioactive DNA-specific probes can be bound to the fragments. The result is a pattern of bands-the DNA fingerprint. One of the first applications of this technique was in forensic science. The uniqueness of a DNA fingerprint can be used for various purposes, such as to convict or exonerate a rape suspect or to prove or disprove paternity. Since the Fist submission of DNA fingerprints as evidence in late 1987, more than a dozen such court cases have followed, and many more cases have yet to go to trial. Because trial evidence is subject to strict guidelines, acceptance of new kinds of evidence can be a slow process. Nevertheless, more and more courts are allowing DNA fingerprints to be introduced as evidence, and widespread forensic use of DNA fingerprints is expected within the next few years. Currently three companies, Cellmark (Germantown, MD), Lifecodes (Valhalla, NY), and Forensic Science Associates (Richmond, CA), perform DNA fingerprinting for forensic purposes. The typical procedure for analyzing a sperm sample from a rape case begins with extraction of DNA from the sample matrix, explains Robin Cotton, manager of research and development at Cellmark. Sperm cells are often collected along with a variety of other cells. Because these sperm cells have many sulfhydryl bonds and are thus resistant to lysing, the sample is treated to lyse the nonsperm cells and digest any proteins present. Once the sperm cells are isolated, the DNA is extracted. Restriction enzymes are then used to cut the DNA into frag-
ments, which are separated by gel electrophoresis. The fragments are transferred to a nylon membrane, the membrane is dried, and the DNA is permanently mounted on the membrane by exposure to UV light. The membrane is then hybridized using a “cocktail” of four probes, each of which is a single locus probe that recognizes two bands. The bands come from each chromosome pair, resulting in a pattern of eight bands. The probes are then removed from the membrane. At this point, each probe is added individually. “We want to know what the allele frequency of the two bands is for the first probe, for the second probe, and so forth,” explains Cotton. “These allele frequencies are then used to determine the probability of someone in a population having all eight of these bands.” How likely is it that two people will
have the same DNA fingerprint? Xver a world population of more than 5 billion, the possibility of finding two identical DNA fingerprints is unlikely. With such evidence, law enforcement officials believe that DNA fingerprinting can be as useful as conventional fingerprints or perhaps even more so, because conventional fingerprints are found at only a fraction of all crime scenes. But courtrooms aren’t the only place where DNA fingerprints are being used. Anthropologists have used DNA fingerprints to investigate the similarity between cultures living continents apart. For example, a genetic defect commonly found among American In-
ANALYTICAL CHEMISTRY. VOL. EO, NO. 22, NOVEMBER 15, 1988
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ANALYTICAL CHEMISTRY, VOL. 60. NO. 22. NOVEMBER 15, 1988
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dians WE founc ong New Guineans from East Asia, lending support to the theory of continental drift. Jeffreys and his colleagues are also studying the DNA fingerprints of large families. By examining the similarities in the minisatellite regions, they hope to discover the genetic defects responsible for certain hereditary diseases. DNA amplification Frequently researchers obtain only a fraction of a single DNA molecule, which is less than the amount of genetic material required for routine DNA analysis. In the past, it would have been impossible to work with such a sample. However, in recent years a technique for amplifying DNA-the polymerase chain reaction (PCR)-has enabled researchers to overcome this obstacle. The PCR begins with the use of primers, which are short sequences of DNA that are complementary to the DNA on each side of the target region. When the sample is heated, the two strands of the sample DNA separate. After a cooling step, the primers bind to their complement, one primer to each strand. At this point, the addition of a DNA polymerase allows the synthesis of two new strands that are complementary to the original two. This cycle is repeated many times, and the resulting amplification allows analysis of the targeted DNA fragment. In the original procedure, developed by Kary Mullis and colleagues at Cetus Corp. (Z),a polymerase fromE. coli was used. Because the activity of the polymerase is lost during the heating required to separate the DNA strands, new polymerase must he added after every cycle. A new procedure using a polymerase from Thermus aquaticus, commonly called the Taq polymerase, allows higher temperatures to be used in the amplification procedure, resulting in a higher level of sensitivity. The usefulness of DNA amplification has been noted by Haig Kazazian of The John Hopkins School of Medicine, who uses the PCR to study the genetic defects that lead to certain inherited anemias. "We are using the PCR exclusively," says Kazazian. "In the last year, we've gone from indirect detection using the Southern blot about 95% of the time to complete direct detection using PCR followed by either restriction digestion of the product, dot hybridization with allelespecific oligonucleotides, or nucleotide sequencing. If we don't find the mutation we're looking for by the first two methods, then we will sequence.'' The PCR procedure has also been used for forensic work by Forensic Sci-
ence Associates. The advantage of this approach is that only a fragment of DNA is required. For example, it is now possible to positively identify a person from the DNA contained in a single hair. The disadvantage of the procedure, according to Cotton, is that the results are not as statistically certain as they are with the nonamplification procedure because the entire DNA molecule isn't used. DNA amplification has even been used to study the biochemistry of healing (3).Daniel A. Rappolee and his colleagues at the University of California at San Francisco and a t Cetus Corp. took macrophages from a mouse wound. After removing the RNA from the macrophages, they converted the RNA to DNA and then used the PCR technique for amplification. Using radioactive probes, they discovered that the macrophages were producing transforming growth factor alpha (TGF-alpha), which is suspected to play an important role in healing. This example is significant because it is the first time that scientists have been able to directly detect the growth factor producta made by macrophages in an open wound.
Another application of DNA amplification is in the examination of DNA that is thousands of years old. Such genetic material is often degraded, thus limiting the amount of DNA that is available for analysis. But the PCR procedure overcomes this difficulty by allowing the small amount of intact DNA to be examined. Scientists have used the PCR technique to study DNA from prehistoric animals. By comparing the results of these studies with DNA from modern animals, evolutionary links can he established. The introduction of an automated PCR instrument developed by Cetus and Perkin-Elmer has had a big impact on the speed of analysis and also on the size of the DNA segment that can he amplified. (Cetus Corp., which holds the patent on the PCR, will gain financially from every product made that uses the technique.) Manual amplification is difficult for DNA fragments much larger than 200 base pairs, but Kazazian and his colleagues have used the automated instrument to amplify segments with as many as 2000 bases. The larger segments allow for more useful cloning and analysis of DNA variability, says Kazazian. He believes
that PCR of even larger segments is possible. More lo come DNA fingerprinting and DNA amplification are not the only techniques generating a great deal of excitement. 0ther areas of interest involve the development of site-specific probes, such as those used to identify the DNA fragments on an electrophoretic gel. Probes have also been designed to identify the presence of oncogenes, which are the genes often implicated in cancer. DNA sequencing (see the REPORT by Lloyd Smith in the March 15,1988,issue) can be used to produce novel hormones, pharmaceuticals, and genetically enpineered organisms. Clearly, DNA analysis is not just the realm of the biochemist anymore. These days, techniques of DNA analysis are expanding the frontiers of science.
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