NR AOUY
AU Knoll,M.; Buck,H.; Engel-Stephan,I.; Eberle,W.
TI Automated Sample Purification and Detection of Sheep Prion Protein Polymorphisms at Codons 136, 154 and 171
QU International Conference - Prion diseases: from basic research to intervention concepts - TSE-Forum, 08.10.-10.10.2003, Gasteig, München - Poster session - DG-02
PT Konferenz-Poster
AB Natural Scrapie seems to be associated with polymorphisms at three codons within the sheep prion protein (PrP). Genotyping of these codons (136,154,171) enables breeding of sheep flocks with low or even no susceptibility for Scrapie. Reliable genotyping of sheep is a major issue since Scrapie seems to develop to a serious problem in several countries. To address this, we developed a combined system for sample preparation and detection of codon 136,154,171 polymorphisms. Deoxyribonucleic acid (DNA) of whole blood samples of sheep with known PrP nucleotide sequence at codons 136,154,171 was purified using Roche MagNA Pure instrument. Detection was performed using Roche LightCycler (LC) instrument in two steps: (1) amplification of a PrP-specific PCR product and (2) detection of polymorphisms 136,154,171 by hybridization of oligo nucleotides (LightCycler HybProbe). LightCycler genotyping of each sheep DNA sample tested was matched with the sequence information showing the reliability of this genotyping assay. In addition the combination of MagNA Pure and LightCycler (PCR Workflow System) offers several advantages for genotyping sheep: (1) fast and automated DNA purification (2) automated pipetting of PCR setups directly into LightCycler capillaries (3) fast analytical procedure (4) minimal human error factor, e. g. sample tube handling, pipetting or cross contamination.
AD M. Knoll, H. Buck, I. Engel-Stephan, W. Eberle, Roche Diagnostics GmbH, Germany
SP englisch
PO Deutschland