Acquisition of a real-time PCR conversion System to Enhance Genetics Research capabilities at the Western South Dakota DNA Core Facility

Real-time PCR is a powerful new technology that will enhance research capabilities at the Western South Dakota DNA Core Facility. This NRI award will be used to purchase an Opticon 1 conversion system to convert an existing thermal cycler to a real-time polymerase chain reaction (PCR) thermal cycler. One project that will benefit from this will be to develop real-time PCR genotyping techniques to aid the management of public and private herds of American bison in order to maintain genetic diversity and identify bison-cattle hybrids. Real-time PCR will also be used to identify alleles of the bison PrP gene for which little information is available. The ability to distinguish between cattle and bison alleles of this gene will be useful in the study of prion diseases. Information about this locus would be extremely informative considering the extent of hybridization of bison with cattle and that it is not known how hybridization events might influence a change in infectivity of prions. Another project that will benefit is a study of the evolutionary relationships of South Dakota walleye populations with previously studied populations using DNA sequence analysis of the mtDNA control region. This has been problematic due to heteroplasmy. Real-time PCR offers a way to screen individual fish for heteroplasmy and also to quantify the mtDNA genomes in individual fish. This will allow the study of intraspecific phylogenies of walleye populations and assess the possible significance of heterogeneity of heteroplasmy frequencies among populations of walleye.
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An Opticon 1 continuous fluorescence detector, and 96-well Alpha Unit (MJ Research) will be purchased to convert an existing DNA Engine thermal cycler chasis (MJ Research) to a real-time PCR thermal cycler. Using existing bison and cattle microsatellite, RFLP and mtDNA marker information, real-time PCR will be used to detect slight sequence differences in the melting curve profiles of alleles of various markers. The profiles of each marker tested will be evaluated for the ability to accurately and efficiently distinguish between bison and cattle alleles. This information will then be used to develop a rapid and cost-effective method to identify bison-cattle hybrids. The PrP gene (the gene involved in transmissible spongiform encephalopathies) of American bison will be cloned and sequenced from our existing bison genomic lambda phage library. Blood and/or tissue samples from numerous individual bison will be collected using standard methods. Sequence information will be used to develop primers that can be used in real-time PCR to amplify various regions of the PrP gene in order to identify different alleles of this gene in the bison population. Samples with different melting curve profiles will then be sequenced in order to compare sequences of the various bison alleles. This information will also be used to compare bison and cattle PrP alleles in order to determine the amount of identity and similarity between the cattle and bison PrP genes. Real-time PCR will be used to amplify the mtDNA d-loop region of walleye, Stizostedion vitreum. Differences between the melting curve profiles of the PCR products will indicate sequence differences in this region and allow quantification of the different mtDNA genomes of individual fish. Combined with traditional DNA sequencing of the different PCR products, the information obtained will then be used to determine the frequency of heteroplasmy within and among South Dakota populations of walleye.
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The objective of this project was to acquire real-time PCR capabilities to enhance the ability of the Western South Dakota DNA Core Facility at Black Hills State University to conduct genomics research. The award, in the amount of $24,490, was used toward the purchase of an Applied Biosystems 7500 Real-Time PCR System. Due to unforeseen delay in the construction of the laboratory in which the equipment was to be housed, the purchase of this equipment did not occur until the fourth quarter of the grant year, December 2004. As a result, the associated projects were also delayed. Two research projects were described in the original proposal that would benefit from real-time PCR capabilities. Project 1: Utilize real-time PCR techniques to develop rapid genotyping methods for genetic loci important to bison management and conservation with initial focus on the bison PrP gene. Preliminary data was obtained prior to the purchase of the equipment. By mid August 2005, exon 3 of the PrP gene, which contains the complete coding region for the prion protein, was sequenced in both directions for 25 individual American bison blood samples given to us by Custer State Park and a private rancher. Two alleles were identified that differed at only one position of the coding region. The same alleles are found in domestic cattle and are, therefore, not useful from a management perspective. However, we have also sequenced a portion of the PrP promoter region in bison, which has been shown to be correlated with resistance in domestic cattle when a 23 base pair insertion is present. None of the bison samples sequenced to date contain this insertion. We are currently looking into the feasibility of using real-time PCR techniques to distinguish between the two different alleles of the promoter region of domestic cattle so that we can quickly screen numerous bison samples for those alleles. Project 2: Develop a real-time PCR method to screen populations of walleye, Stizostedion vitreum, for heteroplasmy and quantify the mtDNA genomes in individual fish in order to study intraspecific phylogenies of walleye populations. Due to the delay in purchasing the equipment this project has also been delayed. Preliminary data has been gathered by sequencing the d-loop region of walleye and noting areas of variability that can be used to develop probes specific for use in real-time PCR experiments. One of the most significant results of this funding is the development of new research projects by BHSU faculty that utilize real-time PCR technology. Such projects include assessing the expression of genes involved in resistance to predation by insect herbivores in species of Arabis, especially those related to glucosinolate production; quantification of alternatively spliced gene products belonging to a novel member of a gene family in tomato; and development of an assay to quantify the levels of expression of stress genes in the endangered minnow, Notropis topeka, in order to gain information on the stress factors present in existing populations so that management strategies can be developed to enhance recovery efforts.
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The acquisition of an Applied Biosystems 7500 Real-Time PCR system by the Western South Dakota DNA Core Facility at Black Hills State University will significantly enhance genomics research capabilities of the faculty at this institution. Several new collaborative projects have begun that will utilize this equipment including, identification of alleles of the prion protein gene associated with resistance or susceptibility to Bovine Spongiform Encephalopathy in bison; a study of gene expression by species of the plant Arabis involved in resistance to insect herbivores; a study of the expression of stress genes in remaining populations of the endangered minnow, Notropis topeka; and quantification of alternatively spliced gene products belonging to a novel member of a gene family in tomato. This equipment will also allow the core facility to offer more thorough services to potential collaborators at other state institutions and agencies.