Basic Genetics and the Australian Shepherd Part Three: DNA Fingerprinting of Canines First printed in the Aussie Times by George P. Johnson Until very recently, the words fingerprints and fingerprinting brought to mind images of detective novels, television police dramas, ink-stained fingers and searches of the FBI's files to find guilty individuals. More and more often though, these words bring to mind images of lab technicians in goggles and white coats peering at dark spots on photographs and speaking words that sound like gibberish to normal people. While the methodologies and terminologies behind each image are completely different, both kinds of "fingerprinting" are used to achieve the same purpose: to provide a means of identification that is individually unique. In the 1890s, it was realized that the patterns of depressions and ridges on the tips of the fingers varied between individuals. Further study determined that these patterns (loops, whorls and arches) were unique to each individual and would provide a means of individual identification. Use of these "fingerprints" became a routine tool for identification because no two individuals had the same pattern, not even identical twins. In the 1990s, DNA "fingerprinting" has become a routine and powerful tool of analysis and finds such varied uses as paternity and maternity determination, identification and analysis of remains from accidents and crime scenes (such as the last Czar of Russia and his family), matching suspects to evidence, and identification of illegally obtained and smuggled wildlife. Unlike actual fingerprints, identical twins would have exactly the same DNA fingerprint; but, because of the great amount of variation possible, fraternal twins would not. What is DNA? DNA or deoxyribonucleic acid, the source of hereditary information for all living things, contains the instructions for making and running individuals throughout their lifetimes. When complexed with proteins, the DNA-protein association makes up the chromosomes. Dogs have 78 chromosomes while humans have 46. The chromosomes are located in the cell's control center, the nucleus. Chemically, DNA is composed of four chemical building blocks (nucleotides) represented here by the abbreviations A, T, G and C. By arranging these blocks in a meaningful sequence, a gene, information is specified to make and run individuals. Genes, therefore, are nothing more than sentences composed of words built from a four-letter alphabet. This is at the same time stupidly simple and elegantly complex because millions of species use the same four-letter alphabet to specify all genetic information. Examples of information specified by a gene would be the black/red coat color or the merle/solid pattern of a dog. Genes are passed via chromosomes to the offspring when sperm and egg fuse during fertilization. At this point, an individual's genetic blueprint is fixed; the fertilized egg begins the process of developing into the new individual by faithfully executing the instructions contained within its DNA. What is a DNA Fingerprint? In addition to the genetic sequences that specify canine traits like coat color and pattern there are sequences whose purpose is not understood at this time. These occur at multiple locations (loci) that are scattered over the chromosomes (throughout the genome) and are highly variable in their composition. One type of variation consists of tandemly arranged repeating units based on just two of the building blocks of DNA; for instance, a sequence of CACA... or, GTGT... Another type of variation consists of tandemly arranged repeating units based on all four of the building blocks of DNA; for instance, a sequence of ATGCATGC... These repeating sequences are known as microsatellites and the number of repeats (alleles) within the sequence varies between individuals. There are other types of microsatellites that differ from the types mentioned here but they follow the same general pattern as the two and four nucleotide pattern which are the types most widely used for fingerprinting. A DNA fingerprint is a profile or listing of the kind of microsatellite present in an individual at each locus (marker) and the allelic composition (number of repeats) for each microsatellite. This is analogous to a listing of the genes and alleles for a red-factored blue merle, i.e. BbMm. The type of microsatellite corresponds to a gene and the number of repeats corresponds to the alleles of that gene; i.e. CA4, where CA indicates the kind of microsatellite and 4 indicates that there are four repeats of the CA sequence (CACACACA). Just as with the alleles for color (B for black and b for red), these alleles are inherited from the parents and can be traced back to them. Barring some rare change or mutation, all of the alleles present at each locus in an individual can be traced back to the parents. Because there are multiple alleles known for each locus, and many loci are used, an incredible number of variations are possible and the chances that any two individuals would have the same profile are almost non-existent. How is a DNA Fingerprint Prepared? The DNA fingerprinting process begins by collecting a tissue sample from the dog. Blood draw or check swabs are two commonly used techniques to collect samples for DNA extractions. Because the laboratory process is so precise and sensitive, great care must be taken not to provide a sample with DNA from more than one donor; therefore, hand washing, wearing disposable gloves and using disposable needles or other collection devices is mandatory for valid results. The laboratory analysis utilizes a process very similar to a cells's own mechanism for duplicating DNA; the loci of the chromosomes containing the microsatellites of interest are duplicated by a process known as the polymerase chain reaction (PCR). This process combines all of the materials needed to copy the alleles for each microsatellite in a "test tube" that is manipulated under very stringent conditions; each microsatellite is copied in a separate tube. In one or two hours, millions of copies of only selected microsatellites are produced from DNA from a tissue sample of the donor dog. The dog's DNA serves as a template for the duplication process. Any tissue that contains the nucleus of cells can provide the chromosomes to serve as templates. The PCR process is necessary to insure that enough material is present for detection and analysis. Following the duplication process, the microsatellite alleles are separated in a process called electrophoresis. A sample from each test tube is placed into a separate reservoir in a special medium known as a gel. The gel serves as a microscopic "Swiss cheese" and when an electrical charge is applied to the gel the fragments of duplicated DNA move through the gel according to size (allele length). Small fragments migrate faster and farther than larger fragments and when finished, the gel has a series of DNA fragments arranged in vertical files. The alleles present in each vertical file represent an individual locus or spot in the dog's genome and are recorded. This listing of the genetic makeup for these loci of the individual becomes the DNA fingerprint. By carefully choosing microsatellites that have a large number of alleles (size variations), the possible number of combinations within DNA fingerprints becomes incredibly large. Thus, it would be very unlikely that another dog would have the same DNA fingerprint. Benefits: A DNA Fingerprint Can ... Because every individual is genetically unique (identical twins have never been proven in dogs), and genetic composition does not change through life, a DNA fingerprint becomes a feature that is permanently associated with an individual. In the case of loss, theft or disputed ownership, a DNA fingerprint can confirm an individual's identity. Thus, the dog can be returned to its correct owner. Additionally, the fingerprint can be used to confirm the dog's identity for a health registry and the results of x-rays, eye exams, blood tests, etc., can be permanently associated with the dog. Proof of parentage is one of the most familiar uses for DNA fingerprinting. This provides for unparalleled accuracy in animal registration and, as more and more generations are fingerprinted, a pedigree can be certified with an accuracy unknown today. DNA fingerprinting is required by some animal registries and has replaced blood typing in those registries as a means of individual identification. In the advent of an accidental breeding, the identity of the actual sire can be determined as can be the sires within mixed-sire litters. Also, DNA fingerprinting can verify the identity of semen used in artificial insemination. Does a DNA Fingerprint Prove Parentage? It is impossible to prove parentage by any means, including blood typing or even DNA fingerprinting. Parentage verification works by excluding individuals based on the allelic composition of the parents relative to the offspring; each allele in an offspring has to be present in one parent, and may even be present in both. If an allele cannot be located in either parent, they are not the sire or the dam of that individual. The following example will illustrate how the process is used in a case of a possible mixed-sire litter. A litter of two is whelped, and there is a question as to whether both pups were sired by the same individual. A review of the genes for color, pattern and trim is of no use in determining the correct sire of the pups. The dam is not in question and when fingerprinted was heterozygous at a locus with alleles CA4 & CA5 (these alleles represent repeats of the CA sequence 4 and 5 times, respectively). When checked at the same locus, the potential sires had the following composition: Potential Sire 1 had alleles CA4 & CA6; and, Potential Sire 2 had alleles CA4 & CA7. The possible genotypes of the pups based on a mating between the dam and Potential Sires 1 and 2 are shown in Figures 1 and 2, respectively. Dam (CA4CA5) CA4 allele CA5 allele Potential Sire 1 (CA4CA6) CA4 allele CA4CA4 CA4CA5 CA6 allele CA4CA6 CA5CA6 Figure 1. Genotypes of Dam and Potential Sire 1, and Potential Genotypes of Offspring Dam (CA4CA5) CA4 allele CA5 allele Potential Sire 2 (CA4CA7) CA4 allele CA4CA4 CA4CA5 CA7 allele CA4CA7 CA5CA7 Figure 2. Genotypes of Dam and Potential Sire 2, and Potential Genotypes of Offspring When fingerprinted, the following results were obtained for the pups: Pup A had alleles CA4 & CA6; and, Pup B had alleles CA4 & CA7. For each pup, the CA4 allele can be attributed to the dam because her other allele, CA5, is not present in either pup. Therefore, the CA6 allele in Pup A and the CA7 allele in Pup B must have come from their sire. Since neither sire possesses both the CA6 and CA7 alleles, this is a mixed-sire litter; and, Potential Sire 2 is excluded from parentage of Pup A and Potential Sire 1 is excluded from parentage of Pup B. Just because an individual has not been excluded from parentage does not make that individual the parent. In the previous example it is highly likely that Potential Sire 1 was the actual sire of Pup A and Potential Sire 2 was the actual sire of Pup B because they were the only dogs on the premises with access to the bitch. But, any dog in the breed which matched the actual sire of each pup in allelic composition for that locus could be the sire as well. So how do we eliminate these individuals? Simply by examining other loci. It becomes highly unlikely that a dog would not be the actual sire if there were matches for all markers examined. Depending on which markers are used and the number of possible alleles available at each marker, the use of between seven and ten markers gives a probability of parentage of 99.5% or greater if matches occur at all loci. This may translate into one chance in hundreds of thousands, or millions; clearly more Aussies than there are alive now and for some time to come. Limitations: A DNA Fingerprint Cannot ... The fingerprinting process discussed in this article is designed to provide a unique means of identification. For use by animal registries its primary goals are the identification of a single individual and verification that the parents of record are listed accurately beyond any reasonable doubt. As currently performed, the fingerprinting process is by far the most accurate way to achieve these goals and it surpasses any other technique now available for these purposes. Two things that it cannot do however, are determine if a dog is a carrier of a defective (disease causing) allele, and if a dog is cross-bred. There are a number of methodologies used for examining the DNA of individuals to see if they are the carriers of defective alleles. One of these methods involves the use of DNA fingerprints as described here, but this research requires a great deal of work (read expensive!) before the fact to determine some pattern between the presence of a marker and its variation and the disease-causing allele. Often, many hundreds of markers must be examined before this is accomplished. Once a pattern is determined, a test can be performed to look for this marker and the variation known to be associated with the disease-causing allele. Once examined, a dog can be rated as affected, carrier, or normal (clear). When available, these tests provide a much faster and inexpensive method of detecting carriers than a typical test cross. It is currently thought that the fingerprinting process described here also cannot detect a second or third generation cross-bred individual. It is impossible to go backwards and discover cross-breeds in the pedigree; this applies to within-breed errors as well. However, as work progresses in identifying the variation in the canine genome, it may become possible to detect cross-bred dogs. Some scientific literature indicates that researchers have detected alleles at some markers that seem to be restricted to a single breed. As more breeds are fingerprinted, and as more individuals in each breed are profiled, breed-specific differences may disappear (being due to an initial small and non-representative sample size) or may become well established. Fingerprinting is rather new to pure-bred dog registries and only time will tell what will be discovered. Questions from the Membership One of the purposes of this series of articles is to answer questions from ASCA's membership. If you have a question about something in one of these articles or anything related to the genetics of the Australian Shepherd, contact me at the address below, by phone, or by email. If I don't know the answer I will search until I do. Your question will be answered privately or in an upcoming issue of the Aussie Times. George P. Johnson Department of Biological Sciences Arkansas Tech University Russellville, AR 72801 Phone: 501-968-0312 FAX: 501-964-0837 Email: george.johnson@mail.atu.edu