Parentage Testing Procedures
A DNA profile, which provides alleles sizes for all microsatellite markers, is obtained, and parentage analysis is performed. A variety of sample types can be utilized for routine testing, including blood, hair, semen, buccal swabs and FTA cards. Non-routine sample types include bone, teeth, saliva, dried blood, urine and feces. DNA is extracted from the samples and microsatellite marker analysis begins with the PCR procedure. In this process a computer program compares the DNA profile of the offspring to those of the presumed parents. A parentage analyst reviews the results and sends the final report. If a listed parent or parents are excluded, additional analysis is performed including retest of samples and the possible use of additional DNA markers to confirm the exclusion.
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Detailed DNA Parent Verification Information
For over four decades, parentage verification has been utilized in animal registration programs. Breeder experiences have proven that parentage testing, in combination with well run breeding programs, can ensure accurate pedigrees. This article will explain the basis of the DNA based parentage test performed at the Veterinary Genetics Laboratory (VGL) and address the role of parentage analysis in animal breeding programs.DNA and Microsatellite Structure
Understanding the DNA based parentage test requires a brief explanation of DNA structure. The DNA molecule contains four variations of a chemical structure called a base. These variants are referred to in shorthand as A, C, G and T. Chromosomes are comprised of millions of these four bases arranged in a linear fashion called a DNA strand. The sequence of these four bases at specific sites, or genes, along the chromosomes is what determines the genetic code for each individual animal. Also found throughout animal genomes are specific sequences of bases repeated in a tandem fashion and referred to as microsatellite DNA markers. The most common microsatellite markers contain a two base sequence repeated in tandem. An example of this is a CA sequence repeated ten times: (...TCAGGTCTACACACACACACACACACACATGCTTATGTACT...) The genomes of mammalian species contain thousands of these microsatellite markers.Genetic Variation and DNA Analysis
The number of tandem repeats found at any given microsatellite marker will vary between individual animals just as will physical appearance. Genetic variation at the microsatellite level has been exploited by molecular biologists for a variety of functions. Microsatellite markers are the basis for individual identification, generally in a forensics application, for paternity analysis in humans and for the majority of animal parentage testing done today. The various forms of a given microsatellite that are identified by differences in repeat number are referred to as alleles and are inherited in a Mendelian fashion. An animal may possess only two alleles for a specific microsatellite, one inherited from its dam and the other from its sire. The size of the alleles possessed by an offspring must correspond to those of the presumed parents. With an increase in the number of repeats there is a proportional increase in the allele size. To detect these size variations DNA is isolated from the sample, microsatellites are amplified by the Polymerase Chain Reaction (PCR) and at the same time tagged with a fluorescent dye. The dye-labeled alleles are detected by laser excitation and their size determined by gel electrophoresis and computer analysis.
Parentage Case Scenarios
The typical animal parentage case, as seen with most domestic species, includes a dam, offspring and one or more sires. Generally the identity of the dam is fairly certain. As mentioned earlier, in order to qualify to a set of parents an offspring must possess the same allele sizes as the parents. Outlined below are a few examples of the types of animal parentage cases submitted to the VGL. The numbers represent the allele sizes, which are the number of DNA base repeats, determined by PCR and gel electrophoresis.
I. Typical Parentage Case
One dam and three possible sires. For simplicity only four markers are shown.
In this case the dam and sire 1 qualify as possible parents. Sires 2 and 3 are excluded at several markers without consideration of the dam. The alleles that have excluded sires 2 and 3 as possible parents are shown in bold. For example, at Marker 1 the offspring's DNA type is 86/112 and Sire 2's type is 96/120; there are no alleles in common. Sire 2 and the offspring also have no alleles in common at Marker 4 and Sire 2 is again excluded.II. Mating Exclusion Case
Sire 4, below, is used to illustrate such a case. The dam, offspring and sire 1 are from example I.
The offspring possesses a Marker B 156 allele, a Marker C 204 allele and a Marker D 226 allele that are not possessed by either the dam or sire 4. This data indicates that one of the parents is incorrect. The offspring could have inherited the Marker B 150 allele, the Marker C 202 allele and the Marker D 224 allele from either parent. Therefore, neither parent can be definitively excluded so the mating must be excluded.
In the majority of cases, if all available microsatellite markers are utilized, one parent will be positively excluded from the mating. However, the outcome of a mating exclusion case can change significantly if only one parent is provided. In this case without knowledge of the dam?s genetic contribution, both sire 1 and 4 would appear to qualify as a parent, which demonstrates the importance of providing both a dam and sire in a parentage case.Accuracy of Parentage Analysis
Parentage testing identifies individuals that, due to a specific combination of marker alleles, could qualify as a parent for a particular offspring. Accurate parentage testing requires breeders to identify possible parents since if considering a randomly selected large group of individuals there could be more than one that qualifies as a parent. As an example, human paternity testing was originally developed as a means to verify that a named individual could or could not be the father of a given child. At most it was meant to determine if one of several men could be the father of a child. The same rules hold true for animal parentage testing. A good application for animal parentage testing is verification that the dam is correct and which of the sires on a particular farm are the actual sire.
Finally, it is important to remember that while parentage exclusions are 100% accurate parentage qualifications are not. The accuracy of most animal parentage tests is greater than 99% when both parents are included in the analysis and drops to around 95% when only one parent is included in the analysis. However, this accuracy will decrease when the potential parents are part of a large group of closely related animals. Again, an animal closely related to an actual parent could possess marker alleles that make it appear to be the correct parent. To prevent erroneous parentage qualifications, breeders need to submit samples from all possible parents when first requesting parentage verification. If more than one sire and one dam qualify as parents of an offspring the laboratory can then test with additional DNA markers to sort out the actual parents.
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