Several scientific studies support the claim that natural scrapie is associated with polymorphisms at three codons within the sheep prion protein (PrP) gene. Genotyping of these codons (codon 136 [alanine, valine], 154 [arginine, histidine], and 171 [glutamine, arginine, histidine]) might enable breeding of sheep flocks with high resistance to scrapie. Fast and reliable high-throughput genotyping of sheep is a major issue, since scrapie is a serious problem in several European countries. To address this, an experimental procedure for DNA preparation and PCR-based detection of codon 136, 154, and 171 polymorphisms was established.
DNA was purified from sheep whole blood and solid tissue samples. Prp genotype detection was performed by amplification of a PrP-specific PCR product and subsequent detection of polymorphisms 136, 154, and 171 by hybridization of oligonucleotides (LightCycler® Hybridization Probes) using the LightCycler® 480 Instrument.
Materials and Methods
Prior to DNA preparation, whole blood samples were anticoagulated with K2EDTA and stored at -20°C.
Nerve tissue (medulla oblongata) and muscle tissue samples were taken to genotype dead and slaughtered sheep.
DNA from thawed whole blood samples was prepared using the MagNA Pure LC DNA Isolation Kit – Large Volume (purification protocol ”DNA LVBlood_20_200µl“). 200 µl whole blood were processed. DNA was recovered in 100 µl elution buffer.
DNA from 70 mg nerve or muscle tissue samples were extracted by High Pure PCR Template Preparation Kit. DNA was recovered in 200 µl elution buffer.
Amplification of the sheep PrP gene by PCR
PCR amplification of a 212-bp-fragment of the sheep PrP gene was performed using the LightCycler® 480 Instrument with 384-well plates. The PCR setup was prepared according to the package insert of LightTyper Sheep PrP Gene Mutation Detection Kit (codons 136, 154, and 171). The PCR protocol for amplification is specified in Table 1 and Table 2.
Analysis of genotypes using the LightCycler® 480 Instrument
Following the PCR run, melting-curve analysis was performed according to the LightCycler® 480 Operator’s Manual. The specified polymorphisms within the sheep PrP gene were detected by melting peaks with a defined temperature. The LightCycler® 480 Software automatically matched the melting peaks with the corresponding polymorphisms.
Results and Applications
Melting curve analysis using the LightCycler® 480 Instrument
The LightCycler® 480 melting curve analysis results in peak patterns specific for the specified sheep PrP genotypes (Figures 1–3). Homozygous (Figures 1a, 2a, 3a) and heterozygous polymorphisms (Figures 1b, 2b, 3b, 3c, 3d) for codon 136, codon 154, and codon 171 are shown.
Number of animals tested
For validation of the test format, 8,441 samples of individual sheep of 31 different breeds were genotyped (at codons 136, 154, and 171). Detailed results and number of animals tested are listed in Table 3.
The experimental workflow including HighPure PCR Template Preparation Kit or MagNA Pure LC Instrument, LightCycler® 480 Instrument and LightTyper Sheep PrP Gene Mutation Detection Kit (codons 136, 154, and 171) is an excellent tool for genotyping the sheep prion gene using whole blood or solid tissue samples. The combination of these workflow elements is an easy, fast, and accurate test format for this application.
This article was originally published in Biochemica 3/2007, pages 7-9. ©Springer Medizin Verlag 2007