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Three-step PCR and RFLP Genotyping of the Swine Ryanodine Receptor Gene Using Aged Single Hair Follicles Delivered by General Mail |
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Y. Kim, S. C. Woo, G. C. Song, H. Y. Park, B. S. Im, G. W. Kim |
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| Abstract |
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We have developed a reliable and noninvasive method for swine genotyping of single locus nuclear gene with aged single hair follicles delivered by general mail. The method is based on booster and nested PCR amplification with step-wise increase of primers and dNTPs concentrations followed by restriction endonuclease digestion. To establish this method, the ryanodine receptor (RYR 1) locus which is an economically important trait in swine industry was employed for genotyping experiment. The 3-step PCR amplication method is much less dependent on the quantity and quality of template DNA and produces enough amplification product for the detection on the ethidium bromide-stained gel such as RFLP analysis. A total of 120 pigs were subjected to the RYR 1 genotyping analysis using three-step PCR method which amplified enough quantity of PCR products from the aged single hair follicles for RFLP analysis and genotyping results were identical to the results of the corresponding ethanol-fixed skeletal muscle tissue. This approach will be a great help for porcine breeders and investigators in genotyping of swine. They can receive genotyping results later by simply plucking single hairs of their pigs at farm and sending them in general mail to the diagnostic laboratory which eliminates the inconveniences to collect ear tissue or blood cells from pigs, or the investigator''''''''s need for travel to farms in order to collect fresh hair samples. |
| Keywords:
Three-step (Booster and Nested) PCR Amplification; RFLP Analysis; Aged Hair Follicles; RYR 1 Locus; Genotyping |
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