DNA fingerprinting (Forensic Science)
In 1985, the English geneticist Alec Jeffreys proposed that newly discovered repetitive sequences in DNA (deoxyribonucleic acid) could be used as a form of genetic fingerprint to identify individuals. DNA fingerprinting represents a combination of both molecular biology and population genetics in that in this process, pieces of DNA are examined for the presence of specific markers and the findings are compared against known samples in a database to establish the prevalence of those markers in the general population.
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Types of Genetic Markers (Forensic Science)
The human genome comprises more than 3.2 billion nucleotides, the letters that are responsible for coding for the proteins that make up and carry out bodily functions. The sequences of the human genome that code for these proteins are called genes. Humans are believed to have approximately twenty-five thousand genes. Between these genes are vast stretches of DNA that do not code for proteins. Within these areas are repetitive sequences called variable number of tandem repeats, or VNTRs. One class of VNTRs is made up of the minisatellites, sequences of up to one hundred nucleotides that may be repeated in tandem up to one thousand times. In addition to the minisatellites, microsatellites have been identified. These are also known as short tandem repeats (STRs) or simple sequence repeats (SSRs). Microsatellites are sequences of two to seven nucleotides that may be repeated hundreds of times. An example is the CA repeat (CACACACA) that occurs on average every thirty-thousand base pairs in the human genome.
Every human being has two copies of genetic information in each cell. This represents the genetic information contributed by each of the individual’s parents. Each STR thus exists in two copies. Often, the number of repeats within a specific STR differs in the parents. These differences are called alleles. In a population, a given STR may be polymorphic, meaning that many different forms (or alleles) of the STR...
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Analysis of DNA Fingerprints (Forensic Science)
When DNA fingerprinting was initially developed, analysts examined DNA patterns using a procedure called the Southern blot. In a Southern blot, DNA is extracted from cells and then cut with a special enzyme called a restriction endonuclease. Restriction endonucleases recognize specific sequences of nucleotides in the DNA. When the sequence has been identified, the enzyme makes a cut in the DNA, generating short fragments that may be separated by size through gel electrophoresis. A radioactive probe is then used to identify specific fragments that contain sequences of nucleotides of interest. Initially, analysts accomplished this task by using minisatellites and restriction fragment length polymorphisms (RFLPs). When exposed to photographic film, the radioactive probes revealed patterns in the DNA that could be used to identify evidence.
Soon after DNA fingerprinting began, the entire process was greatly simplified by the invention of the polymerase chain reaction (PCR). Instead of cutting the DNA with restriction enzymes, the analyst copies specific sections of the genome, in this case the area containing the microsatellite repeats, millions of times. The amplified sections are then separated by gel electrophoresis, stained, and photographed. Because the fragments are much smaller than those generated during a Southern blot, the results may be ready in just a few hours of time. As with a Southern blot, the length...
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Further Reading (Forensic Science)
Butler, John M. Forensic DNA Typing: Biology, Technology, and Genetics of STR Markers. 2d ed. Burlington, Mass.: Elsevier Academic Press, 2005. Provides a detailed examination of DNA fingerprinting analysis using STR markers. Intended for readers with background in the sciences.
Jeffreys, A. J., V. Wilson, and S. L. Thein. “Individual-Specific ’Fingerprints’ of Human DNA.” Nature 316 (1985): 76-79. Landmark paper that introduced the concept of DNA fingerprinting as a method of identification.
Kobilinsky, Lawrence F., Louis Levine, and Henrietta Margolis-Nunno. Forensic DNA Analysis. New York: Chelsea House, 2007. Presents a comprehensive introduction to the use of STRs in DNA fingerprinting. Includes discussion of future directions, including mitochondrial and Y chromosome analyses.
Rudin, Norah, and Keith Inman. An Introduction to Forensic DNA Analysis. 2d ed. Boca Raton, Fla.: CRC Press, 2002. Provides a good introduction to the use of biological evidence in forensics as well as the history and application of DNA fingerprinting in forensic investigations.
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Genetic Differences Among Individuals (Genetics & Inherited Conditions)
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 that 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...
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The First DNA Fingerprints (Genetics & Inherited Conditions)
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.
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Variable Number Tandem Repeat Loci (Genetics & Inherited Conditions)
The type of sequence Jeffreys used 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 1 to 6 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.
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The Polymerase Chain Reaction (Genetics & Inherited Conditions)
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 depend 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,...
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Current Approaches (Genetics & Inherited Conditions)
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.
Analyzing genes from cellular DNA can be limited if biological samples are limited. This occurred with many victims from the World Trade Center terrorist attack. Extensive burning and decomposition of victims found months later resulted in little biological tissue for genetic testing. Cells contain two copies of every gene, but cells contain thousands of mitochondria (organelles that provide energy for cells) that have their own DNA. Mitochondrial DNA is a source of more DNA analysis where the biological tissue supply is severely limited....
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Human ForensicForensic geneticsDNA fingerprinting and Paternity Testing (Genetics & Inherited Conditions)
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.
For validity concerns, it is important to consider false positives and false negatives. A false-positive genetic test identifies a genetic match when none exists, whereas a false-negative genetic test declares no match when a genetic match actually exists. How can false positives and false negatives occur in genetic testing? One way relates to the laboratory mechanics of genetic testing. Electrophoresis is used in genetic testing. Electrophoresis separates genetic fragments along a path that is around 8 inches in length. If a genetic fragment travels the exact same distance as another genetic fragment—the two genetic fragments are...
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Other Uses for VNTR Genotyping (Genetics & Inherited Conditions)
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.
VNTR typing has been used to study the epidemiology of disease transmission. A 2008 study published in Tuberculosis genotyped forty-one Mycobacterium tuberculosis isolates from the Warao people, an indigenous population with a high tuberculosis (TB) incidence living in a geographically isolated area in Venezuela. This genetic study showed that 78 percent of the TB strains were in clusters, suggesting a very high transmission rate. VNTR typing is a useful tool to study the molecular epidemiology of tuberculosis, and this type of genetic analysis promises to yield more valuable information in the treatment and prevention of disease.
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Further Reading (Genetics & Inherited Conditions)
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 Speciments. 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.
Maes, M., et al. “24-Locus MIRU-VNTR Genotyping Is a Useful Tool to Study the Molecular Epidemiology of Tuberculosis Among Warao Amerindians in Venezuela.” Tuberculosis 88, no. 5 (2008): 490-494. A study that shows the value of DNA fingerprinting.
Rudin, Norah, and...
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Web Sites of Interest (Genetics & Inherited Conditions)
DNA Fingerprinting, Genetics and Crime: DNA Testing and the Courtroom. http://www.fathom.com/course/21701758/index.html. An online “seminar” from the University of Michigan explaining principles, procedures, and issues related to DNA fingerprinting.
Human Genome Project. http://www.ornl.gov/sci/techresources/Human_Genome/elsi/gmfood.shtml. Comprehensive Web site with information on the Human Genome Project, medicine and genetics, ethical, legal and social issues, and educational resources.
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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