A robust, low- to medium-throughput prnp genotyping system in sheep.
BACKGROUND
In many countries breeding programs for resistance to scrapie in sheep are established. Therefore, the demand on genotyping capacities of the polymorphisms of the prion protein gene (prnp) relevant to presently known disease associations and EU regulations is steadily increasing. Most published typing methods are not well suited for routine typing of large sample numbers in smaller service laboratories for different reasons: they require partly manual data processing, sophisticated and sensitive protocols, high efforts regarding time and manpower, multiple step reactions or substantial hardware investments. To overcome these drawbacks, we developed a prnp typing method that is based on a 'multiplex amplification refractory mutation system' (ARMS) reaction.


METHODS
In this study we combined the amplification refractory mutation system (ARMS) with standard fluorescent based fragment length analyses method to develop a prnp genotyping method (PRNP ARMS).


RESULTS
By optimised primer design it was possible to type the 4 relevant single nucleotide polymorphisms (SNPs) in the prnp simultaneously in one multiplex reaction. Automated fragment length analysis enabled automated allele designation. Suitability of the PRNP ARMS for routine application was proven by typing samples with known genotypes and larger sample numbers from half-sib families.


CONCLUSIONS
The ARMS PRNP typing method established in this study is universally suited for a broad range of typing projects with different requirements. It provides an efficient and inexpensive diagnostic mutation analysis that will improve the quality of prnp genotyping compared with other low-cost methods. It can be implemented by most molecular genetic laboratories using standard equipment.
Buitkamp
Johannes
J
Bavarian State Research Center for Agriculture, Institute of Animal Breeding, Prof,-Dürrwaechter-Platz 1, 85586 Poing, Germany. johannes.buitkamp@lfl.bayern.de
Semmer
Jördis
J
D000483:Alleles; D000818:Animals; D004247:DNA / Q000737:chemistry; D004252:DNA Mutational Analysis / Q000379:methods / Q000662:veterinary; D005787:Gene Frequency; D005838:Genotype; D016133:Polymerase Chain Reaction / Q000662:veterinary; D020641:Polymorphism, Single Nucleotide; D018620:PrPSc Proteins / Q000737:chemistry / Q000235:genetics*; D012608:Scrapie / Q000175:diagnosis* / Q000235:genetics; D012756:Sheep
D018620:PrPSc Proteins; D004247:DNA