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Encyclopedia of Genetics, Rev. Ed. DNA Fingerprinting Field of Study: Human genetics and social issues Significance: DNA fingerprinting includes a variety of techniques in which individuals are uniquely identified through examination of specific DNA sequences which are expected to vary widely among individuals. Uses for these technologies include not only practical applications in forensic analysis and paternity tests but also basic research in paternity, breeding systems, and ecological genetics for many nonhuman species. Key Terms MICROSATELLITE: a type of VNTR in which the repeated motif is 1 to 6 base pairs; synonyms include simple sequence repeat (SSR) and short tandem repeat (STR) MINISATELLITE: a type of VNTR in which the repeated motif is 12 to 500 hundred base pairs in length POLYMERASE CHAIN REACTION (PCR): a laboratory procedure for making millions of identical copies of a short DNA sequence VARIABLE NUMBER TANDEM REPEAT (VNTR): a type of DNA sequence in which a short sequence is repeated over and over; chromosomes from different individuals frequently have different numbers of the basic repeat, and if many of these variants are known, the sequence is termed a hypervariable Genetic Differences Among Individuals All individuals, with the exception of twins and other clones, are genetically unique. Theoretically it is therefore possible to use these genetic differences, in the form of DNA sequences, to identify individuals or link samples of blood, hair, and other features to a single individual. In practice, individuals of the same species typically share the vast majority of their DNA sequences; in humans, for example, well over 99 percent of all of the DNA is identical. For individual identification, this poses a problem: Most of the sequences that might be examined are identical (or nearly so) among randomly selected individuals. The solution to this problem is to focus only on the small regions of the DNA which are known to vary widely among individuals. These regions, termed hypervariable, are typically based on repeat sequences in the DNA. Imagine a simple DNA base sequence, such AAC (adenine-adenine-cytosine), which is repeated at a particular place (or locus) on a human chromosome. One chromosome may have eleven of these AAC repeats, while another might have twelve or thirteen, and so on. If one could count the number of repeats on each chromosome, it would be possible to specify a diploid genotype for this chromosomal locus: An individual might have one chromosome with twelve repeats, and the other with fifteen. If there are many different chromosomal variants in the population, most individuals will have different genotypes. This is the conceptual basis for most DNA fingerprinting. DNA fingerprint data allow researchers or investigators to exclude certain individuals: If, for instance, a blood sample does not match an individual, that individual is excluded from further consideration. However, if a sample and an individual match, this is not proof that the sample came from that individual; other individuals might have the same genoytpe. If a second locus is examined, it becomes less likely that two individuals will share the same genotype. In practice, investigators use enough independent loci that it is extremely unlikely that two individuals will have the same genotypes over all of the loci, making it possible to identify individuals within a degree of probability expressed as a percentage, and very high percentages are possible. The First DNA Fingerprints Alec Jeffreys, at the University of Leicester in England, produced the first DNA fingerprints in the mid-1980's. His method examined a twelve-base sequence that was repeated one right after another, at many different loci in the human genome. Once collected from an individual, the DNA was cut using restriction enzymes to create DNA fragments that contained the repeat sequences. If the twelve-base sequence was represented by more repeats, the fragment containing it was that much longer. Jeffreys used agarose gel electrophoresis to separate his fragments by size, and he then used a specialized staining technique to view only the fragments containing the twelve-base repeat. For two samples from the same individual, each fragment, appearing as a band on the gel, should match. This method was used successfully in a highly publicized rape and murder case in England, both to exonerate one suspect and to incriminate the perpetrator. While very successful, this method had certain drawbacks. First, a relatively large quantity of DNA was required for each sample, and results were most reliable when each sample compared was run on the same gel. This meant that small samples, such as individual hairs or tiny blood stains, could not be used, and also that it was difficult to store DNA fingerprints for use in future investigations. Variable Number Tandem Repeat Loci The type of sequence Jeffreys exploited is now included in the category of variable number tandem repeats (VNTRs). This type of DNA sequence is characterized, as the name implies, by a DNA sequence which is repeated, one copy right after another, at a particular locus on a chromosome. Chromosomes vary in the number of repeats present. VNTRs are often subcategorized based on the length of the repeated sequence. Minisatellites, like the Jeffreys repeat, include repeat units ranging from about twelve to several hundred bases in length. The total length of the tandemly repeated sequences may be several hundred to several thousand bases. Many different examples have since been discovered, and they occur in virtually all eukaryotes. In fact, the Jeffreys repeat first discovered in humans was found to occur in a wide variety of other species. Shorter repeat sequences, typically one to six bases in length, were subsequently termed microsatellites. In humans, AC (adenine-cytosine) and AT (adenine-thymine) repeats are most common; an estimate for the number of AC repeat loci derived from the Human Genome Project suggests between eighty thousand and ninety thousand different AC repeat loci spread across the genome. Every eukaryote studied to date has had large numbers of microsatellite loci, but they are much less common in prokaryotes. The Polymerase Chain Reaction The development of the polymerase chain reaction (PCR) in the mid-1980's, and its widespread use and optimization in DNA labs a few years later offered an alternative approach to DNA fingerprinting. The PCR technique makes millions of copies of short segments of DNA, with the chromosomal location of the fragments produced under the precise control of the investigator. PCR is extremely powerful and can be used with extremely small amounts of DNA. Because the fragments amplified are small, PCR can also be used on partially degraded samples. The size and chromosomal location of the fragments produced depends on the DNA primers used in the reaction. These are short, single-stranded DNA molecules that are complementary to sequences that flank the region to be amplified. With this approach, an investigator must find and determine the DNA sequence of a region containing a VNTR. Primers are designed to amplify the VTNR region, together with some flanking DNA sequences on both ends. The fragments produced in the reaction are then separated by length using gel electrophoresis so that differences in length, attributable to different numbers of the repeat, become apparent. For a dinucleotide repeat like AC, fragments representing different numbers of repeats, and hence different alleles, differ by a multiple of two. For instance, a researcher might survey a number of individuals and find fragments of 120, 122, 124, 128, and 130 base pairs in length. Current Approaches Most current approaches to DNA fingerprinting use data collected simultaneously from a number of different VNTR loci, most commonly microsatellites. Preferably, the loci are PCR amplified using primers with fluorescent dyes attached, so that fragments from different loci are uniquely tagged with different colors. The fragments are then loaded in polyacrylamide DNA gels of the type used for DNA sequencing and separated by size. The fluorescent colors and sizes of the fragments are determined automatically, using the same automated machines typically used for DNA sequencing. DNA fingerprint data generated in this way are easily stored and saved for future comparisons. Since each allelic variant is represented by a specific DNA fragment length, and because these are measured very precisely, the initial constraint of running samples for comparison on the same gel is avoided. Human Forensic and Paternity Testing Although several different systems have been developed and used, a widely employed current standard comprises the Federal Bureau of Investigation's Combined DNA Index System (CODIS), with thirteen core loci. These thirteen are tetranucleotide (TCTA) microsatellite repeat loci, located on autosomes. Each locus has many known alleles, in some cases more than forty; the genetic variation is well characterized, and databases of variation within a variety of ethnic groups are available. In addition to its role in criminal cases, this technique has seen widespread use to establish or exclude paternity, in immigration law to prove relatedness, and to identify the remains of casualties resulting from military combat and large disasters. Other Uses for VNTR Genotyping Soon after VNTRs were discovered in humans and used for DNA fingerprinting, researchers demonstrated that the same or similar types of sequences were found in all animals, plants, and other eukaryotes. The method pioneered by Jeffreys was, only a few years later, used for studies of paternity in wild bird populations. Since then, microsatellite analysis has come to dominate studies of relatedness, paternity, breeding systems, and other questions of individual identification in wild species of all kinds, including plants, insects, fungi, and vertebrates. Researchers now know, for example, that among the majority of birds which appear monogamous, between 10 and 15 percent of all progeny are fathered by males other than the recognized mate. Paul R. Cabe See AlsoCriminality; Forensic Genetics; Genetic Testing; Genetics, Historical Development of; Human Genetics; Paternity Tests; Repetitive DNA. Further Reading Burke, Terry, R. Wolf, G. Dolf, and A. Jeffreys, eds. DNA Fingerprinting: Approaches and Applications. Boston: Birkhauser, 2001. Describes repetitive DNA and the broad variety of practical applications to law, medicine, politics, policy, and more. Aimed at the layperson. Fridell, Ron. DNA Fingerprinting: The Ultimate Identity. New York: Scholastic, 2001. The history of the technique, from its discovery to early uses. Aimed at younger readers and nonspecialists. Herrmann, Bernd, and Susanne Hummel, eds. Ancient DNA: Recovery and Analysis of Genetic Material from Paleographic, Archaeological, Museum, Medical, and Forensic Specimens. New York: Springer-Verlag, 1994. Written when DNA fingerprinting was just coming to the fore and films such as Jurassic Park were in theaters, this collection of papers by first-generation researchers reflects the broad applications of the technology, including paleontological investigations. Hummel, Susanne. Fingerprinting the Past: Research on Highly Degraded DNA and Its Applications New York: Springer-Verlag, 2002. Manual about typing ancient DNA. Rudin, Norah, and Keith Inman. An Introduction to Forensic DNA Analysis. Boca Raton, Fla.: CRC Press, 2002. An overview of many DNA typing techniques, along with numerous examples and a discussion of legal implications. Wambaugh, Joseph. The Blooding. New York: Bantam Books, 1989. The policeman-turned-writer offers a fascinating account of the British rape and murder case in which DNA fingerprinting was first used. Web Site of Interest Earl's Forensic Page. http://members.aol.com/EarlNMeyer/DNA.html. Summarizes how DNA fingerprinting works and its use in crime investigations and in determining paternity. Iowa State University Extension and Office of Biotechnology, DNA Fingerprinting in Agricultural Genetics Programs. http://www.biotech.iastate.edu/biotech_info_series. Site links to a comprehensive and illustrative article on the role of DNA fingerprinting in agriculture. |
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