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Development of a SNP Assay for the Differentiation of Allelic Variations in the mdx Dystrophic Mouse Model
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The purpose of this study was to develop a SNaPshotÂ® assay to simultaneously discriminate between the dystrophic and wild type (wt) alleles in mdx mice. The mdx mouse is an animal model for Duchenne muscular dystrophy (DMD), a severe and fatal muscle wasting disease. To evaluate possible treatments and to carry out genetic studies, it is essential to distinguish between mice that carry the mutant dystrophic or wt allele(s). The current Amplification-Resistant Mutation System (ARMS) assay used to genotype mdx mice is labor intensive and sometimes fails to yield typing results, which reduce its efficiency as a screening tool. An alternative assay based on single nucleotide polymorphism (SNP) extension technology (i.e., SNaPshotÂ®) would be advantageous because its specificity and capability to be automated would reduce the labor involved and increase the fidelity of each assay. A SNaPshotÂ® assay has been developed that provides a robust and potentially automatable assay that discriminates between the wt and dystrophic alleles. The assay has been optimized to use: an undiluted DNA in the PCR, a 0.1 ÂµM PCR primer concentration, a full PCR product for the SNP extension reaction, a 50ÂºC annealing temperature for the SNP extension in accordance with standard SNaPshotÂ® conditions, and a 0.4 ÂµM concentration of the SNP extension primer. The advantages of the resultant SNaPshotÂ® assay over the ARMS assay include higher fidelity, robustness, and more consistent performance within and among laboratories, and reduced risk of human error.
- Masters Theses